Extracellular vesicles for agent delivery

ABSTRACT

The present invention relates to the field of extracellular vesicles. More specifically, the present invention provides methods and compositions for using extracellular vesicles as a vector for nucleic acid treatment in vivo of various diseases. In a specific embodiment, the present invention provides an extracellular vesicle isolated from a cell comprising one or more microRNAs (miRNAs) that have been loaded ex vivo into the vesicle so that the miRNAs are present in a higher concentration than when measured in the same extracellular vesicle isolated directly from the cell. In another embodiment, the present invention provides a method for treating cholangiocarcinoma in a subject comprising the step of administering to the subject a plurality of exosomes comprising miR- 195.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/545,937, filed Jul. 24, 2017, which is the U.S. national phaseapplication, pursuant to 35 U.S.C. § 371, of PCT InternationalApplication Ser. No.: PCT/US2016/015791, filed Jan. 29, 2016,designating the United States and published in English, which claimspriority to and the benefit of U.S. Provisional Application No.62/109,764, filed Jan. 30, 2015, and U.S. Provisional Application No.62/150,318, filed Apr. 21, 2015, each of which are incorporated hereinby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Oct. 5, 2018, is named167689_011203_US_SL.txt and is 1,452 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of extracellular vesicles(e.g., exosomes, microvesicles, macrovesicles). More specifically, thepresent invention provides compositions comprising extracellularvesicles for delivery of agents (e.g., polynucleotides, polypeptides,small molecules) and methods of using such compositions, for example, intherapeutic, imaging, and research methods.

BACKGROUND OF THE INVENTION

Cholangiocarcinoma (CCA) is the second most common primary liver cancerin the United States. The survival of CCA patients is dismal, usuallymeasured in months. Primary therapy with surgery is applicable to fewerthan 20% of patients. Photodynamic therapy and chemotherapy provideresponses in a minority of patients without curative intent. Thus thereis an urgent need for improved treatment for CCA, and novel treatmentmodalities for CCA are potentially translatable to other types ofcancer. In addition, there exists a need for methods that selectivelydeliver therapeutics to cancer cells. Such compositions and methodscould be translated to a wide array of disease treatments.

SUMMARY OF THE INVENTION

The present invention provides extracellular vesicles (EVs) derived froma cancer associated cell (e.g., fibroblast-like cell, stromal cell)comprising an agent (e.g., polypeptide, polynucleotide, small molecule),and methods of using such EVs to deliver the agent to a target cell.

The invention generally provides an extracellular vesicle isolated froma cancer associated fibroblast (CAF), where the vesicle contains anexogenous agent.

In one aspect, the invention provides an extracellular vesicle isolatedfrom a cancer associated fibroblast (CAF), where the vesicle contains aheterologous polynucleotide identified as being down-regulated in theCAF, and where the extracellular vesicle selectively targets a cancercell.

In various embodiments of the above-aspects or any other aspect of theinvention delineated herein, the agent is an exogenous polynucleotide.In various embodiments of the above-aspects the polynucleotide ismiR-195, miR-126, or miR-192 or is a polynucleotide encoding miR-195,miR-126, or miR-192. In various embodiments of the above-aspects thepolynucleotide is a vector encoding miR-195, miR-126, or miR-192. Invarious embodiments of the above-aspects, the polypeptide is arecombinant polypeptide heterologously expressed in the CAF or loadedinto the cell or extracellular vesicle ex vivo. In various embodimentsof the above-aspects, the polynucleotide is a recombinant polynucleotidethat is heterologously expressed in the cell or is loaded into the cellex vivo. In various embodiments of the above-aspects, the recombinantpolynucleotide is a microRNA. In various embodiments of theabove-aspects the microRNA is miR-195, miR-126, or miR-192. In variousembodiments of the above-aspects, the small molecule is a lipid or otherhydrophobic small molecule. In various embodiments of the above-aspects,the small molecule is doxorubicin, cisplatin, or phosphatidylethanolamine. In various embodiments of the above-aspects, thephosphatidyl ethanolamine is derivatized with an agent selected from thegroup consisting of rhodamine, fluorescein, biotin, streptavidin, asmall molecule, a polynucleotide, and a polypeptide. In variousembodiments of the above-aspects, the polypeptide is an antibody, apolypeptide that localizes to a specific cell type, a therapeuticprotein, or protein that can be used for imaging purposes. In variousembodiments of the above-aspects, the agent is a nanoparticle,paramagnetic particle, microsphere, or nanosphere for magnetic imaging.In various embodiments of the above-aspects, the cancer associatedfibroblast is a stromal cell. In various embodiments of theabove-aspects, the stromal cell is derived from a tumormicroenvironment. In various embodiments of the above-aspects, the tumoris a cholangiocarcinoma, hepatocellular carcinoma, or hepatoma. Invarious embodiments of the above-aspects, the tumor is a breast cancertumor, pancreatic tumor, glioblastoma, melanoma, lung cancer tumor,ovarian cancer tumor, or any other type of cancer. In variousembodiments of the above-aspects, the extracellular vesicle is isolatedfrom a bodily fluid selected from the group consisting of blood, plasma,serum, urine, stool, semen, cerebrospinal fluid, prostate fluid,lymphatic drainage, bile fluid, and pancreatic secretions. In variousembodiments of the above-aspects, the extracellular vesicle is isolatedfrom cell culture media. In various embodiments of the above-aspects,the extracellular vesicle is isolated from cells cultured in conditionedmedia obtained from a culture containing cancer cells. In variousembodiments of the above-aspects, the extracellular vesicle is isolatedfrom a culture containing a CAF derived from a fibroblast,fibroblast-like cell, stellate cell, or myofibroblast. In variousembodiments of the above-aspects, the CAF expresses one or more of alphasmooth muscle actin and/or collagen. In various embodiments of theabove-aspects, the fibroblast-like cell has a fibroblast morphology. Invarious embodiments of the above-aspects, the vesicle expressesincreased levels of one or more markers selected from the groupconsisting of alpha-SMA, Collagen, Vimentin (FSP-1), S100,Metalloproteinases, NG2, PDGFR-B, SDF1/CXCL12, CD34, Fibroblastactivation protein (FAP), FSP-1, CD31, Thy-1, and Gremlin. In variousembodiments of the above-aspects, the vesicle expresses reduced levelsof laminin. In various embodiments of the above-aspects, the CAF isderived from a fibroblast cultured for at least 1-14 days in thepresence of a cancer cell or in the presence of conditioned mediaderived from a cancer cell culture. In various embodiments of theabove-aspects, the vesicle is isolated from mammalian cells. In variousembodiments of the above-aspects, the vesicle is an exosome or amicrovesicle.

In another aspect, the invention provides a method for obtaining anextracellular vesicle, the method involving culturing a fibroblast orstromal cell in conditioned media obtained from a cancer cell culture,and isolating extracellular vesicles from the media.

In another aspect, the invention provides an extracellular vesicleproduced according to the method of the above aspects.

In another aspect, the invention provides a pharmaceutical compositioncontaining a vesicle of any of the above aspects.

In another aspect, the invention provides a method of delivering anagent to a cell, the method involving contacting the cell with a vesicleof any of the above-aspects, thereby delivering the agent to the cell.

In another aspect, the invention provides a method of reducing a tumorin a subject, the method involving contacting the cell with the vesicleof any of the above aspects.

In another aspect, the invention provides a method of altering geneexpression in a cell, the method involving contacting the cell with avesicle of any previous aspect.

In another aspect, the invention provides a method for treating cancerin a subject comprising administering to the subject a pharmaceuticalcomposition comprising an effective amount of the vesicle of anyprevious aspect.

In another aspect, the invention provides a method for treatingcholangiocarcinoma, hepatocellular carcinoma, or hepatoma in a subjectcomprising administering to the subject a pharmaceutical compositioncomprising an effective amount of an extracellular vesicle isolated froma CAF over-expressing a recombinant polynucleotide encoding miR-195,miR-192, or miR-126.

In another aspect, the invention provides a pharmaceutical compositioncomprising a first and a second extracellular vesicle, where eachvesicle contains a different agent. In one embodiment, each vesiclecomprises a different miRNA.

In another aspect, the invention provides a pharmaceutical compositioncomprising a plurality of exosomes, where each exosome contains one ofmiR-195, miR-192, or miR-126.

In another aspect, the invention provides a composition for imagingstudies, the composition comprising an extracellular vesicle isolatedfrom a cancer associated fibroblast (CAF) or fibroblast-like cell, wherethe vesicle contains a detectable agent. In one embodiment, thedetectable agent is an imaging agent. In another embodiment, the imagingagent is a nanoparticle, magnetite, nanoparticle, paramagnetic particle,microsphere, nanosphere, and is selectively targeted to cancer cells.

In another aspect, the invention provides a kit for delivering an agentto a cell the kit comprising an extracellular vesicle isolated from acancer associated fibroblast (CAF) or fibroblast-like cell, where thevesicle contains an agent.

In various embodiments of the above-aspects, the method inhibits tumorcell proliferation. In various embodiments of the above-aspects, theextracellular vesicle is an exosome. In various embodiments of theabove-aspects, the cancer cells are derived from a liver cancer orbreast cancer. In various embodiments of the above-aspects, the cell iscultured for between about 3-days and 2 weeks in conditioned media. Invarious embodiments of the above-aspects, the method further containsincubating the isolated extracellular vesicle in a solution comprisingan agent. In various embodiments of the above-aspects, the extracellularvesicle is incubated for between about 1 and 4 hours. In variousembodiments of the above-aspects, the fibroblast or stromal cellcontains a vector encoding a recombinant protein or microRNA. In variousembodiments of the above-aspects, the extracellular vesicle contains anincreased level of a recombinant protein, polynucleotide, or smallmolecule than a corresponding control cell not cultured in conditionedmedia. In various embodiments of the above-aspects, the extracellularvesicle is a microvesicle.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “cancer associated fibroblast (CAF)” is meant a fibroblast thatexpresses increased levels of alpha-smooth muscle actin (SMA),PDGFRbeta, and/or collagen relative to a control fibroblast. In oneembodiment, a CAF expresses at least about 2-fold, 5-fold, 10-fold morealpha-SMA, PDGFRbeta, and collagen relative to a non-CAF fibroblast(i.e., a fibroblast derived from healthy non-cancerous tissue, or thathas not been cultured in conditioned media derived from cancer cells). ACAF derived EV promotes tumor growth and metastasis. In contrast, CAFsof the invention comprise agents that inhibit tumor growth. In anotherembodiment, a CAF expresses reduced levels of miR-195, miR-192 andmiR-126 relative to a reference. In another embodiment, a CAFoverexpresses any one or more of the following markers: Actin (a-SMA),Collagen, Vimentin (FSP-1), S100, Metalloproteinases, NG2, PDGFR-B, SDF1(CXCL12), CD34, Fibroblast activation protein (FAP) and FSP-1 (as wellas CD31), Thy-1, and Gremlin relative to a reference. In anotherembodiment, a CAF expresses reduced levels of laminin relative to areference. In addition to stromal cells, CAFs may be derived from cellshaving proximity to the tumor in vivo. Thus, CAFs may be derived fromcells associated with blood vessels or local deposits of fat near theterm. In some instances, a CAF is identified at a site distant from thetumor. Such CAFs are identified as CAFs or their subtypes by markingstudies. In particular embodiments, a cancer associated cell (CAC) maybe used in place of a CAF. CACs include brain derived glia,oligodendroglia, and microglia. Other CACs include Breast-EMT and bonemarrow stem cells which have become CAFs. Other cells useful in theinvention include reactive cell populations associated with cancer thatexpress in various proportions FSP-1, S100, Metalloproteinases, NG2a-SMA, and PDGFR-B.

As used herein, the term “microRNA,” “miRNA,” or “miR” refers to RNAsthat function post-transcriptionally to regulator expression of genes,usually typically by binding to complementary sequences in the threeprime (3′) untranslated regions (3′ UTRs) of target messenger RNA (mRNA)transcripts, usually resulting in gene silencing. miRNAs are typicallysmall regulatory RNA molecules, for example, 21 or 22 nucleotides long.The terms “microRNA,” “miRNA,” and “miR” are used interchangeably.

By “miR-195” is meant a polynucleotide or fragment thereof having atleast about 85% or greater nucleic acid sequence identity to thepolynucleotide sequence provided at NCBI Accession No. NR_029712 that iscapable of modulating gene expression. In one embodiment, the miRNAaffects the stability and/or translation of mRNAs.

An exemplary miR-195 nucleic acid sequence is provided below:

Homo sapiens miR-195 (SEQ ID NO: 1)  1agcttccctg gctctagcag cacagaaata ttggcacagg gaagcgagtc tgccaatatt 61ggctgtgctg ctccaggcag ggtggtgThe exemplary sequence represents the predicted microRNA stem-loop. Somesequence at the 5′ and 3′ ends may not be included in the intermediateprecursor miRNA produced by Drosha cleavage.

By “miR-195 gene” is meant the polynucleotide sequence encoding themiR-195 miRNA.

By “miR-192” is meant a polynucleotide or fragment there of having atleast about 85% or greater identity to the polynucleotide sequenceprovided at NCBI Accession No. NR_029578 that is capable of modulatinggene expression. In one embodiment, the miRNA affects the stabilityand/or translation of mRNAs. An exemplary miR-192 nucleotide sequence isprovided below:

Homo sapiens miR-192 (SEQ ID NO: 2)  1gccgagaccg agtgcacagg gctctgacct atgaattgac agccagtgct ctcgtctccc 61ctctggctgc caattccata ggtcacaggt atgttcgcct caatgccagcThe exemplary sequence represents the predicted microRNA stem-loop. Somesequence at the 5′ and 3′ ends may not be included in the intermediateprecursor miRNA produced by Drosha cleavage.

By “miR-192 gene” is meant the polynucleotide sequence encoding themiR-192 miRNA.

By “miR-126” is meant a polynucleotide or fragment there of having atleast about 85% or greater identity to the polynucleotide sequenceprovided at NCBI Accession No. NR_029695 that is capable of modulatinggene expression. In one embodiment, the miRNA affects the stabilityand/or translation of mRNAs. An exemplary miR-126 nucleotide sequence isprovided below:

Homo sapiens miR-126 (SEQ ID NO: 3)  1cgctggcgac gggacattat tacttttggt acgcgctgtg acacttcaaa ctcgtaccgt 61gagtaataat gcgccgtcca cggcaThe exemplary sequence represents the predicted microRNA stem-loop. Somesequence at the 5′ and 3′ ends may not be included in the intermediateprecursor miRNA produced by Drosha cleavage.

By “miR-126 gene” is meant the polynucleotide sequence encoding themiR-126 miRNA.

By “agent” is meant a polypeptide, polynucleotide, or fragment, oranalog thereof, small molecule, or other biologically active molecule.

By “alteration” is meant a change (increase or decrease) in theexpression levels of a gene or polypeptide as detected by standard artknown methods such as those described above. As used herein, analteration includes a 10% change in expression levels, preferably a 25%change, more preferably a 40% change, and most preferably a 50% orgreater change in expression levels.

As used herein, the term “animal” refers to any member of the animalkingdom. The term “animal” may refer to humans at any stage ofdevelopment or any non-human animal at any stage of development. In someembodiments, the term “animal” may refer to a transgenic or geneticallyengineered animal or a clone.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Methods of preparingantibodies are well known to those of ordinary skill in the science ofimmunology. Antibodies can be intact immunoglobulins derived fromnatural sources or from recombinant sources and can be immunoreactiveportions of intact immunoglobulins. Antibodies are typically tetramersof immunoglobulin molecules. Tetramers may be naturally occurring orreconstructed from single chain antibodies or antibody fragments.Antibodies also include dimers that may be naturally occurring orconstructed from single chain antibodies or antibody fragments. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab′) 2, as well as single chain antibodies (scFv),humanized antibodies, and human antibodies (Harlow et al., 1999, In:Using Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual,Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426). In someembodiments, the antibody specifically binds to C4A polypeptide.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′) 2, and Fv fragments, linear antibodies, scFvantibodies, single-domain antibodies, such as camelid antibodies(Riechmann, 1999, Journal of Immunological Methods 231:25-38), composedof either a VL or a VH domain which exhibit sufficient affinity for thetarget, and multispecific antibodies formed from antibody fragments. Theantibody fragment also includes a human antibody or a humanized antibodyor a portion of a human antibody or a humanized antibody.

As used herein, the term “approximately” or “about,” as applied to oneor more values of interest, refers to a value that is similar to astated reference value. In some embodiments, the term “approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, or less in either direction of the stated reference value unlessotherwise stated or otherwise evident from the context.

By “control” is meant a standard or reference condition. The term“control” refers to a standard against which results are compared. Insome embodiments, a control is used at the same time as a test variableor subject to provide a comparison. In some embodiments, a control is ahistorical control that has been performed previously, a result oramount that has been previously known, or an otherwise existing record.A control may be a positive or negative control.

By “decreases” is meant a reduction by at least about 5% relative to areference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, oreven by as much as 75%, 85%, 95% or more.

By “an effective amount” is meant the amount of an agent required toameliorate the symptoms of a disease relative to an untreated patient.In one embodiment, the disease is cancer (e.g., cholangiocarcinoma,hepatocellular carcinoma, hepatoma). In other embodiments, the diseaseis a single gene disorder including, but not limited to, cysticfibrosis, sickle cell anemia, Tay-Sachs disease, myotonic dystrophy,Duchenne muscular dystrophy, Fragile X syndrome, glycogen storagediseases, and spinal muscular atrophy. As would be appreciated by one ofordinary skill in the art, the exact amount required to treat a diseasewill vary from subject to subject, depending on age, general conditionof the subject, the severity of the condition being treated, theparticular compound and/or composition administered, and the like. Theeffective amount of active agent(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “exogenous” is meant foreign. An exogenous agent is one that is notnaturally occurring in the cell, such as a protein that is recombinantlyexpressed.

As used herein, the term “exosome” refers to a small membraneextracellular vesicle of ˜30-300 nm diameter that is secreted fromproducing cells into the extracellular environment, as describedinitially by Trams et al., 1981, BBA. The surface of an exosomecomprises a lipid bilayer from the membrane of the donor cell, and thelumen of the exosome is topologically the same as the cytosol from thecell that produces the exosome. The exosome contains proteins, RNAs,lipids, and carbohydrates of the producing cell, though some may bemodified or added to the exosome after its release from the cell, eitherthrough natural processes or by experimental manipulation.

As used herein, the term “exosome” refers to a small membraneextracellular vesicle of ˜30-300 nm diameter that is secreted fromproducing cells into the extracellular environment, as describedinitially by Trams et al., 1981, BBA. The surface of an exosomecomprises a lipid bilayer from the membrane of the donor cell, and thelumen of the exosome is topologically the same as the cytosol from thecell that produces the exosome. The exosome contains proteins, RNAs,lipids, and carbohydrates of the producing cell, though some may bemodified or added to the exosome after its release from the cell, eitherthrough natural processes or by experimental manipulation.

By “fragment” is meant a portion (e.g., at least 10, 25, 50, 100, 125,150, 200, 250, 300, 350, 400, or 500 amino acids or nucleic acids) of aprotein or nucleic acid molecule that is substantially identical to areference protein or nucleic acid and retains the biological activity ofthe reference.

By “heterologous” is meant originating in a different cell type orspecies from the recipient.

A “host cell” is any prokaryotic or eukaryotic cell that contains eithera cloning vector or an expression vector. This term also includes thoseprokaryotic or eukaryotic cells that have been genetically engineered tocontain the cloned gene(s) in the chromosome or genome of the host cell.

By “inhibits a neoplasia” is meant decreases the propensity of a cell todevelop into a neoplasia or slows, decreases, or stabilizes the growthor proliferation of a neoplasia.

As used herein, the term “in vitro” refers to events or experiments thatoccur in an artificial environment, e.g., in a petri dish, test tube,cell culture, etc., rather than within a multicellular organism.

As used herein, the term “in vivo” refers to events or experiments thatoccur within a multicellular organism.

As used herein, the term “isolated” refers to a substance, molecule, orentity that has been either separated from at least some of thecomponents with which it was associated when initially produced innature or through an experiment, and/or produced, prepared, ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about98%, about 99%, substantially 100%, or 100% of the other components withwhich they were initially associated. In some embodiments, isolatedagents are more than about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, substantially 100%, or 100% pure. As used herein, a substanceis “pure” if it is substantially free of other components.

By “inhibitory nucleic acid molecule” is meant a single stranded ordouble-stranded RNA, siRNA (short interfering RNA), shRNA (short hairpinRNA), or antisense RNA, or a portion thereof, or an analog or mimeticthereof, that when administered to a mammalian cell results in adecrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in theexpression of a target sequence. Such inhibitory nucleic acid moleculesmay delivered using compositions of the invention. Typically, a nucleicacid inhibitor comprises or corresponds to at least a portion of atarget nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “modification” is meant any biochemical or other synthetic alterationof a nucleotide, amino acid, or other agent relative to a naturallyoccurring reference agent.

By “neoplasia” is meant any disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancer is a neoplasia. Examples ofcancers include, without limitation, leukemias (e.g., acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, cholangiocarcinoma (also termed bile ductcarcinoma), choriocarcinoma, seminoma, embryonal carcinoma, Wilm'stumor, cervical cancer, uterine cancer, testicular cancer, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, andretinoblastoma). Lymphoproliferative disorders are also considered to beproliferative diseases.

In some embodiments, “cancer” can include histologic and molecularsubtypes of liver cancer, pancreatic cancer, prostate cancer, breastcancer, hepatocellular carcinoma, colon cancer, lung cancer, lymphoma,leukemia, melanoma, basal cell cancer, cervical cancer, colorectalcancer, stomach cancer, bladder cancer, anal cancer, bone cancer, braintumor, esophageal cancer, gall bladder cancer, gastric cancer,testicular cancer, Hodgkin Lymphoma, intraocular melanoma, kidneycancer, oral cancer, melanoma, neuroblastoma, Non-Hodgkin Lymphoma,ovarian cancer, retinoblastoma, skin cancer, throat cancer, and thyroidcancer. Fibroblasts having proximity to any of the aforementioned cancertypes or grown in a culture comprising such cancer cells are CAFs. Forexample, breast cancer associated fibroblasts are those growing in aculture that also comprises a cancer cell. Cholangiocarcinoma orhepatocellular cancer associated fibroblasts are those growing in aculture that also comprises a cancer cell.

As used herein, the term “microvesicle” refers to a single membranevesicle secreted by cells that may have a larger diameter than thosewhich some refer to as exosomes. Microvesicles may have a diameter (orlargest dimension where the particle is not spheroid) of between about10 nm to about 5000 nm (e.g., between about 50 nm and 1500 nm, betweenabout 75 nm and 1500 nm, between about 75 nm and 1250 nm, between about50 nm and 1250 nm, between about 30 nm and 1000 nm, between about 50 nmand 1000 nm, between about 100 nm and 1000 nm, between about 50 nm and750 nm, etc.). Microvesicles suitable for use in the present inventionoriginate from cells yet different subpopulations of microvesicles mayexhibit different surface/lipid characteristics. Alternative names formicrovesicles include, but are not limited to, exosomes, ectosomes,membrane particles, exosome-like particles, and apoptotic vesicles. Asused herein, an abbreviated form “MV” is sometime used to refer tomicrovesicle.

As used herein, the term “microvesicle” refers to a membranous particlecomprising fragments of plasma membrane that is derived from variouscell types. Typically, microvesicles have a diameter (or largestdimension where the particle is not spheroid) of between about 10 nm toabout 5000 nm (e.g., between about 50 nm and 1500 nm, between about 75nm and 1500 nm, between about 75 nm and 1250 nm, between about 50 nm and1250 nm, between about 30 nm and 1000 nm, between about 50 nm and 1000nm, between about 100 nm and 1000 nm, between about 50 nm and 750 nm,etc.). Typically, at least part of the membrane of the microvesicle isdirectly obtained from a cell (also known as a donor cell).Microvesicles suitable for use in the present invention may originatefrom cells by membrane inversion, exocytosis, shedding, blebbing, and/orbudding. Depending on the manner of generation (e.g., membraneinversion, exocytosis, shedding, or budding), the microvesiclescontemplated herein may exhibit different surface/lipid characteristics.

Alternative names for microvesicles include, but are not limited to,exosomes, ectosomses, membrane particles, exosome-like particles, andapoptotic vesicles. As used herein, an abbreviated form “MV” is sometimeused to refer to microvesicle.

As used herein, an individual “suffering from” a disease, disorder, orcondition means that the person has been diagnosed with or displays oneor more symptoms of the disease, disorder, or condition

By “nucleic acid molecule” is meant an oligomer or polymer ofribonucleic acid or deoxyribonucleic acid, or analog thereof. This termincludes oligomers consisting of naturally occurring bases, sugars, andintersugar (backbone) linkages as well as oligomers having non-naturallyoccurring portions which function similarly. Such modified orsubstituted oligonucleotides are often preferred over native formsbecause of properties such as, for example, enhanced stability in thepresence of nucleases. In certain embodiments, the term “nucleic acidmolecule” refers to genetic material that can be transferred via EVsincluding, but not limited to, miRNA, mRNA, tRNA, rRNA, siRNA, shRNA,DNA (including fragments, plasmids, and the like). Such geneticmaterials can be transferred to EVs via transfection, transformation,electroporation, and microinjection.

By “obtaining” as in “obtaining the inhibitory nucleic acid molecule” ismeant synthesizing, purchasing, or otherwise acquiring the inhibitorynucleic acid molecule.

By “operably linked” is meant that a first polynucleotide is positionedadjacent to a second polynucleotide that directs transcription of thefirst polynucleotide when appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the second polynucleotide.

By “positioned for expression” is meant that the polynucleotide of theinvention (e.g., a DNA molecule) is positioned adjacent to a DNAsequence that directs transcription and translation of the sequence(i.e., facilitates the production of, for example, a recombinantmicroRNA molecule described herein).

By “portion” is meant a fragment of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides.

By “reference” is meant a standard or control condition.

By “reporter gene” is meant a gene encoding a polypeptide whoseexpression may be assayed; such polypeptides include, withoutlimitation, glucuronidase (GUS), luciferase, chloramphenicoltransacetylase (CAT), and beta-galactosidase.

By “selectively deliver” is meant that the majority of the EV isdelivered to a targeted cell type relative to non-target cells presentin the culture, tissue, or organ. In embodiments, greater than about50%, 60%, 70%, 80%, 90%, 95% or even approaching 100% of the EVs aredelivered to a desired cell type. In other embodiments, only about 10%,15%, 20% 25%, 30%, 35%, or 40% of the EVs are delivered to non-targetcells.

The term “siRNA” refers to small interfering RNA; a siRNA is a doublestranded RNA that “corresponds” to or matches a reference or target genesequence. This matching need not be perfect so long as each strand ofthe siRNA is capable of binding to at least a portion of the targetsequence. SiRNA can be used to inhibit gene expression, see for exampleBass, 2001, Nature, 411, 428 429; Elbashir et al., 2001, Nature, 411,494 498; and Zamore et al., Cell 101:25-33 (2000).

“As used herein, the term “stromal cell” refers to non-vascular,non-inflammatory, non-epithelial connective tissue cells of any organthat surround a tumor. Stromal cells are also known as cancer-associatedfibroblasts. Stromal cells support the function of the parenchymal cellsof that organ. Fibroblasts and pericytes are among the most common typesof stromal cells. The stromal cells can be derived from numerous bodytissue types, including, but not limited to, breast tissue, thymictissue, bone marrow tissue, bone tissue, dermal tissue, muscle tissue,respiratory tract tissue, gastrointestinal tract tissue, genitourinarytissue, central nervous system tissue, peripheral nervous system tissue,reproductive tract tissue.

As used herein, the term “subject” refers to a human or any non-humananimal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horseor primate). A human includes pre and post natal forms. In manyembodiments, a subject is a human being. A subject can be a patient,which refers to a human presenting to a medical provider for diagnosisor treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

The term “pharmaceutically-acceptable excipient” as used herein meansone or more compatible solid or liquid filler, diluents or encapsulatingsubstances that are suitable for administration into a human.

By “specifically binds” is meant a molecule (e.g., peptide,polynucleotide) that recognizes and binds a protein or nucleic acidmolecule of the invention, but which does not substantially recognizeand bind other molecules in a sample, for example, a biological sample,which naturally includes a protein of the invention.

By “substantially identical” is meant a protein or nucleic acid moleculeexhibiting at least 50% identity to a reference amino acid sequence (forexample, any one of the amino acid sequences described herein) ornucleic acid sequence (for example, any one of the nucleic acidsequences described herein). Preferably, such a sequence is at least60%, more preferably 80% or 85%, and still more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “targets” is meant alters the biological activity of a targetpolypeptide or nucleic acid molecule.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, apolynucleotide molecule encoding (as used herein) a protein of theinvention.

By “vector” is meant a nucleic acid molecule, for example, a plasmid,cosmid, or bacteriophage, that is capable of replication in a host cell.In one embodiment, a vector is an expression vector that is a nucleicacid construct, generated recombinantly or synthetically, bearing aseries of specified nucleic acid elements that enable transcription of anucleic acid molecule in a host cell. Typically, expression is placedunder the control of certain regulatory elements, including constitutiveor inducible promoters, tissue-preferred regulatory elements, andenhancers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. An image of a co-culture of cancer cells and the fibroblast-like(e.g. fibroblasts, stellate cells, etc.) cell, LX-2 (. HuCCT1 CCA cellsare marked with EGFP and LX2 fibroblasts are unstained.

FIG. 2. Table showing downregulation of multiple miRs in thefibroblast-like LX2 following their co-culture with CCA cells. The tablepresents the Ct value and the qRT-PCR value normalized to U6. The ratioof qRT-PCR expression in LX2 cells cultured alone (control) or in thepresence of cancer cells (F/CAFs) is highlighted in the right column.

FIG. 3. Restoration of miR-195 in the LX-2 fibroblast-like cell issufficient to inhibit invasiveness of co-cultured cancer cells. Fourdifferent human and rat CCA cells were co-cultured with LX2-NSM orLX2-miR-195 cells. Invading cells were visualized by Crystal Violetstaining.

FIG. 4. Up-regulation of miR-195 in fibroblast-like cells inhibitsco-cultured cancer cells that were permitted to exchange media, but werenot in direct contact. From left to right, the slides demonstratedecreased invasion, migration and growth of cancer cells induced bymediators released in media by LX2-195 cells vs. LX2-control.

FIG. 5. LX2-miR-195 fibroblast-like cells release soluble factors thatcause elevated levels of miR-195 in cancer cells. Levels of miR-195 weremeasured in three different CCA cancer cells following their exposure tosoluble factors from either (left bars) LX2 fibroblasts or (right bars)LX2-195 cells that overexpress miR-195.

FIG. 6. LX2-miR-195 cells secrete ˜60-fold higher levels of miR-195 inexosomes/EVs than control LX2 cells.

FIG. 7. EVs derived from a hepatic fibroblast-like cell are targeted toCCA cancer cells in vivo., and selectively deliver a protein cargo tothe cancer cells but not to surrounding parts of the liver or to otherorgans of the body. EVs (indicated by the anti-mCherry staining in thefigure from the presence of the expression of the TSG101/mCherry fusionprotein which is expressed in cells and secreted to) are selectivelyenriched in pockets of the tumor (DAPI stain), that are surrounded bythe endogenous fibroblasts (indicated by the α-SMA staining for activefibroblast). EVs are visualized by staining for an EV cargo protein thatwas expressed in the fibroblast like cells, demonstrating selectivedelivery of protein to cancer cells in vivo.

FIG. 8. EV-carried plasmid designed to express Cre recombinase isselectively delivered to rats via tail vein injections. The tumor areawas stained with antibodies to detect alpha-SMA (a marker of activatedfibroblasts), DAPI (nuclear stain, which detects all cells), and alsovisualized to detect GFP, which is only expressed if the introduced EVsdelivered Cre-expressing DNA into the CCA cells. CCA cells that did nottake up functional Cre remained red in these experiments . We observedcords of fibroblasts (stained with anti-alpha SMA), as well as pocketsof cancer cells, many of which were expressing GFP, establishingselective delivery of DNA into the cancer cells in vivo.

FIG. 9. miR-195-loaded EVs inhibit CCA growth in vivo. EVs were loadedwith (left panels) a non-specific miR mimic or (right panels) a miR-195mimic and injected into rats with CCA. 30 days later, the rats weresacrificed. Tumors were significantly smaller in animals that had beeninjected with miR-195-loaded EVs.

FIG. 10. miR-195-loaded EVs inhibit CCA tumor growth, as measured byvolume (left graph), as well as weight (right graph). The tumorsresected from rats were measured and weighed. The first 3 bars (frontthe left) in each graph represent 3 tumors from rats treated with thenegative control (EVs-NSM), while the 3 bars on the right in each graphrepresent 3 tumors from rats treated with EVs-miR-195.

FIG. 11. miR-195 downregulates CDK6 and VEGF when directly transfectedinto BDEneu cells (left panel), when conditioned media from LX2 cells(treated with miR-195 or NSM) is utilized (middle panel), and whentreated with exosomes loaded with miR-195 vs. NSM (right panel).

FIG. 12. Tail vein treatment of CCA with EVs-miR-195 increases thesurvival in rats by 50% vs. control.

FIG. 13. LX2 cells expressing miR-126 inhibit CCA invasiveness in vitro.HuCCT1 cells were co-cultured directly with LX2 cells expressing either(upper image) a control miR, or (lower image) miR-126. Invasiveness ofHuCCT1 cells was decreased 3.2 fold when co-cultured with LX2-126 cells.

FIG. 14. LX2 cells expressing miR-126 inhibit CCA migration 4-fold invitro. HuCCT1 cells were co-cultured directly with LX2 cells expressingeither a controls miR or miR-126. Migration was measured in a scratchassay.

FIGS. 15A-15C. Mammary fibroblast-derived EVs deliver a small moleculeto breast cancer cells. MDA-MB-231 cells (stably expressing thefluorescent protein tdTomato) were grown in the presence of primaryhuman mammary fibroblast cells that had previously been labeled with afluorescent lipid (N-F-PE; N-fluorescein-phosphatidylethanolamine(Avanti polar lipids)) that is selectively secreted from human cells inEVs (Booth et al., J. Cell Biol. 2006; Fang et al., PLoS Biol. 2007).Over the course of 2-3 days, the (FIG. 15A) tdTomato-expressing humanbreast cancer (seen as white or light signal emitting cells on black andwhite drawings) cells took up the (FIG. 15B) EVs that had been releasedfrom the primary mammary fibroblast cell line. Cells were also stainedwith (FIG. 15C) DAPI to visualize the nucleus.

FIGS. 16A-16D. Mammary fibroblast-derived EVs deliver a protein tobreast cancer cells. MDA-MB-231 cells (stably expressing the fluorescentprotein tdTomato) were grown in the presence of primary human mammaryfibroblast cells that had previously been transfected with a plasmiddesigned to express Acyl-GFP, a form of GFP that is secreted from humancells in EVs. Over the course of 2-3 days, the (FIG. 16A)tdTomato-expressing human breast cancer cells took up the (FIG. 16B) thefluorescent lipid, N-F-PE labeled EVs that had been released from theprimary mammary fibroblast cell line. Cells were also stained with (FIG.16C) DAPI to visualize the nucleus, and (FIG. 16D) the images weremerged to show the presence of CAF-derived EVs in the breast cancer cell(in this case, in the nucleus).

FIGS. 17A-17B. Mammary fibroblasts promote the neoplastic phenotype ofMDA-MB-231 breast cancer cells. MDA-MB-231 cells (stably expressing thefluorescent protein td Tomato) were grown alone or in the presence ofprimary human mammary fibroblast cells. (FIG. 17A) The diameter ofMDA-MB-231 cells grown on their own was approximately 200 relativeunits, but increased ˜7-fold upon co-culture with CAFs, an increase incell size that was apparent as early as 3 hours after co-culture withmammary fibroblasts and was complete within 1 day. Experiments wereperformed in triplicate, followed by calculation of average and standarddeviation. Significant difference from t=1 hr (p, 0.05) were observedfor all but the 2 hr sample. (FIG. 17B) Growth of MDA-MB-231 cells wasinduced ˜2-fold by co-culture with mammary fibroblasts. MDA-MB-231 cellswere plated on culture dishes. The next day, the dishes were either (1)grown on their own, or (2,3) were populated with mammary fibroblast (2)HMF line or (3) MMF line, to a density of ˜20%. The next day the numberof red MDA-MB-231 cells in each dish was counted. Experiments wereperformed in triplicate, and the averages and standard deviations showedsignificant differences between each experimental sample (p<0.05) fromthat of the control cancer cells grown on their own.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides extracellular vesicles (EVs) derived from acancer associated cell (e.g., fibroblast-like cell, stromal cell)comprising an agent (e.g., polypeptide, polynucleotide, small molecule),and methods of using such EVs to deliver the agent to a target cell.

The invention is based, at least in part, on the discovery thatfibroblast gene expression is altered in fibroblasts that grow inproximity to cancer cells (e.g., in stroma) or in conditioned mediawhere cancer cells had previously been cultured. Such cells are termedcancer associated fibroblasts (CAF). As reported in detail below, thegene expression of CAFs is altered following their growth in cancerconditioned media or in stroma. For example, CAFs have increasedexpression of CAF markers: alpha-smooth muscle actin (SMA), PDGFRbeta,and collagen. In one embodiment, a CAF expresses at least about 2-fold,5-fold, 10-fold more alpha-SMA, PDGFRbeta, and collagen relative to anon-CAF fibroblast (i.e., a fibroblast derived from healthynon-cancerous tissue, or that has not been cultured in conditioned mediaderived from cancer cells). We show here that there is also asignificant decrease in multiple miRs, including miR-195, miR-192 andmiR-126. These microRNAs are involved in the transition from normalfibroblasts to CAFs. The overexpression of miR-195 in the CAF reversesmany of the changes observed in not only in CAFs expressing mir-195, butin neighboring cells as well. Surprisingly, this effect was mediated byextracellular vesicles isolated from the mir-195 overexpressing cells.Levels of miR-195 were >60-fold higher in these EVs than in EVs isolatedfrom control cells that were not over-expressing mir-195. In furtherexperiments, cells over-expressing polypeptides and polynucleotides werefound to shed EVs comprising increased levels of the over-expressedpolypeptide or polynucleotide. When injected into rats having CCA, thesefibroblast-derived vesicles were highly enriched within the CCA cellsrelative to non-cancer cells.

Accordingly, the invention provides extracellular vesicles (EVs) derivedfrom CAFs that comprise an agent (e.g., polypeptide, polynucleotide,small molecule), and methods of using such EVs to selectively deliverthe agent to a target cell (e.g., cancer cell) in vivo or in vitro.

Cholangiocarcinoma

Cholangiocarcinoma (CCA) is the second most common primary liver cancer.CCAs are very desmoplastic cancers (similar to pancreatic cancer, andsome breast cancers). As described herein, we identified microRNAspecies that are relatively downregulated in fibroblast-like cells,along the continuum of inactive-to activated-to cancerassociated-fibroblasts (CAFs). Studies in vitro showed that‘therapeutic’ upregulation of these miR species in fibroblast-like cellsresulted in less growth and invasiveness of neighboring cancer cells.Without intending to be bound by theory, it is likely thatcancer-associated fibroblast-like cells play a regulatory role in CCAand other tumors. Thus, we have demonstrated that our therapy interfereswith the signaling between fibroblast-like cells and cancer cells. Theresult is to restrict the growth and invasion of cancer. Inunderstanding this signaling, as described herein, we demonstrated thattransport of extracellular vesicles (EVs) between fibroblast like cellsand cancer cells, in both the CCA model and in a breast cancer model,constitutes a rich signaling network which involves miRNAs and can alsoinvolve the transfer of proteins and lipids. We then engineered such EVsto contain as cargo the desired miR species, the desired protein, or thedesired small molecule. The fibroblast cell-derived EVs are used tointerfere with the signaling network that influences proliferation orinvasion by cancer cells. Results described herein below indicate thatEVs derived from fibroblast-like cells and loaded with microRNAs canaffect the growth and invasion of cancer cells. Moreover, in vivoexperiments demonstrated that EVs loaded with miRs can be systemicallydelivered and then selectively concentrate in liver tumors. Thisdelivery was sufficient to decrease cancer growth and increase theoverall survival (statistically significant) of treated animals. Thesefibroblast-like cell-derived EVs do not accumulate in normal livercells, nor do these EVs accumulate in other tissues (e.g. kidney, lung,etc.).

In conclusion, our studies demonstrate the existence and functioning ofEV exchange between fibroblast-like cells and cancer cells in two cancermodels. We show that miRs loaded into EVs from fibroblast-like cells canhave a functional role in control of the cancer cells. We show that EVsof fibroblast-like cell origin can be loaded with functional miRs, DNAs,proteins, and lipids. In addition, we show that EVs of fibroblast-likecell origin when loaded with miRs selectively target cancer cells invivo and diminish their growth. Finally, EVs of fibroblast-like cellorigin loaded with miRs can be systemically administered to animalsbearing cancers with resulting reduction of tumor growth and resultingsurvival benefit.

Polynucleotides for Delivery

EVs derived EVs containing a microRNA may be used to deliver themicroRNA to a target cell. MicroRNAs (miRNAs) are 20-24 nucleotide RNAmolecules that regulate the stability or translational efficiency oftarget mRNAs. miRNAs have diverse functions including the regulation ofcellular differentiation, proliferation, and apoptosis (Ambros, Nature431, 350-5 (2004)). Although strict tissue- anddevelopmental-stage-specific expression is critical for appropriatemiRNA function, few mammalian transcription factors that regulate miRNAshave been identified.

In general, EVs of the invention comprise a polynucleotide that isdownregulated in a cell of interest (e.g., cancer cell). The EV rescuesthe down regulation by increasing levels of the polynucleotide. In otherembodiments, the EV provides a replacement polynucleotide that replacesor corrects a defective polynucleotide present in the cell.

In one embodiment, an EV derived from a fibroblast-like cell comprises amiR-195, miR-192, or miR-126 microRNA. In another embodiment, EV derivedfrom a fibroblast-like cell comprises a nucleic acid sequence encoding amicroRNA, such as miR from fibroblast-like cells can be used to delivervirtually any polynucleotide, including RNA, DNA, an antisenseoligonucleotide, a short interfering RNA (siRNA), a short hairpin RNA(shRNA), or plasmid DNA polynucleotides and modified oligonucleotides.Exemplary siRNAs include siRNAs targeting Anti-RhoA/C, geranylgeranyl(or farnesyl) and transferase inhibitors of Ras activation,cerivastatin, palbococlib, also siRNA to CXCR4 in breast cancermetastases.

Polynucleotides provided in EVs include Mir -195, miR-192, or miR-126,as well as nucleic acid molecules.

In one embodiment, we have found that CCA cells alter the geneexpression profile of surrounding fibroblasts, including reducedexpression of miR-195; overexpression of miR-195 in CAFs is sufficientto inhibit CCA growth, migration, and invasion in vitro; miR-195 issecreted from CAFs within EVs; elevating miR-195 levels in CAFs issufficient to up-regulate the levels of miR-195 in neighboring cancercells; and intravenous injection of miR-195-loaded EVs inhibit CCAgrowth and extends survival in vivo.

Expression vectors having a polynucleotide with therapeutic function canbe delivered to cells of a subject having a disease (e.g., cancer) usingthe EVs of the invention.

In a specific embodiment, the DNA encodes a protein with a specificfunction, either of diagnostic or therapeutic potential, such as Crerecombinase. In another embodiment, the nucleic acid molecule inhibitsexpression of a tumor suppressor gene as a way to induce a large animalmodel of cancer biology. In a more specific embodiment, the tumorsuppressor gene is p53.

The EV comprising nucleic acid molecules are selectively delivered totarget cells of a subject (e.g., cancer cells) in a form in which theyare taken up and are advantageously expressed so that therapeuticallyeffective levels can be achieved.

An isolated nucleic acid molecule can be manipulated using recombinantDNA techniques well known in the art. Thus, a nucleotide sequencecontained in a vector in which 5′ and 3′ restriction sites are known, orfor which polymerase chain reaction (PCR) primer sequences have beendisclosed, is considered isolated, but a nucleic acid sequence existingin its native state in its natural host is not. An isolated nucleic acidmay be substantially purified, but need not be. For example, a nucleicacid molecule that is isolated within a cloning or expression vector maycomprise only a tiny percentage of the material in the cell in which itresides. Such a nucleic acid is isolated, however, as the term is usedherein, because it can be manipulated using standard techniques known tothose of ordinary skill in the art.

Transducing viral (e.g., retroviral, adenoviral, lentiviral andadeno-associated viral) vectors can be used for somatic cell genetherapy, especially because of their high efficiency of infection andstable integration and expression (see, e.g., Cayouette et al., HumanGene Therapy 8:423-430, 1997; Kido et al., Current Eye Research15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649,1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al.,Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, apolynucleotide can be cloned into a retroviral or other vector andexpression can be driven from its endogenous promoter, from theretroviral long terminal repeat, or from a promoter specific for atarget cell type of interest. Other viral vectors that can be usedinclude, for example, a vaccinia virus, a bovine papilloma virus, or aherpes virus, such as Epstein-Barr Virus (also see, for example, thevectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988;Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990;Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic AcidResearch and Molecular Biology 36:311-322, 1987; Anderson, Science226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al.,Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviralvectors are particularly well developed and have been used in clinicalsettings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson etal., U.S. Pat. No. 5,399,346).

Polynucleotide expression can be directed from any suitable promoter(e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), ormetallothionein promoters), and regulated by any appropriate mammalianregulatory element. For example, if desired, enhancers known topreferentially direct gene expression in specific cell types can be usedto direct the expression of a nucleic acid. The enhancers used caninclude, without limitation, those that are characterized as tissue- orcell-specific enhancers.

EVs derived from fibroblast-like cells can also be used to delivernucleic acid molecules comprising a modified nucleic acid. Nucleic acidmolecules include nucleobase oligomers containing modified backbones ornon-natural internucleoside linkages. Oligomers having modifiedbackbones include those that retain a phosphorus atom in the backboneand those that do not have a phosphorus atom in the backbone. For thepurposes of this specification, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone are alsoconsidered to be nucleobase oligomers. Nucleobase oligomers that havemodified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. Various salts, mixed salts and free acid forms arealso included. Representative United States patents that teach thepreparation of the above phosphorus-containing linkages include, but arenot limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

Nucleobase oligomers having modified oligonucleotide backbones that donot include a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, and CH₂ component parts. Representative UnitedStates patents that teach the preparation of the above oligonucleotidesinclude, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

Nucleobase oligomers may also contain one or more substituted sugarmoieties. Such modifications include 2′-O-methyl and 2′-methoxyethoxymodifications. Another desirable modification is2′-dimethylaminooxyethoxy, 2′-aminopropoxy and 2′-fluoro. Similarmodifications may also be made at other positions on an oligonucleotideor other nucleobase oligomer, particularly the 3′ position of the sugaron the 3′ terminal nucleotide. Nucleobase oligomers may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

In other nucleobase oligomers, both the sugar and the internucleosidelinkage, i.e., the backbone, are replaced with novel groups. Methods formaking and using these nucleobase oligomers are described, for example,in “Peptide Nucleic Acids (PNA): Protocols and Applications” Ed. P. E.Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. RepresentativeUnited States patents that teach the preparation of PNAs include, butare not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262,each of which is herein incorporated by reference. Further teaching ofPNA compounds can be found in Nielsen et al., Science, 1991, 254,1497-1500.

Polypeptide Delivery

The invention provides EVs comprising proteins. In a specificembodiment, the EV-delivered protein corrects a deficiency of the cellor subject, or induces the death of infected or deficient cells.Recombinant polypeptides of the invention are produced using virtuallyany method known to the skilled artisan. Typically, recombinantpolypeptides are produced by transformation of a suitable host cell withall or part of a polypeptide-encoding nucleic acid molecule or fragmentthereof in a suitable expression vehicle.

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. A polypeptide of the invention may be produced in aprokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g.,Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammaliancells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells areavailable from a wide range of sources (e.g., the American Type CultureCollection, Rockland, Md.; also, see, e.g., Ausubel et al., CurrentProtocol in Molecular Biology, New York: John Wiley and Sons, 1997). Themethod of transformation or transfection and the choice of expressionvehicle will depend on the host system selected. Transformation andtransfection methods are described, e.g., in Ausubel et al. (supra);expression vehicles may be chosen from those provided, e.g., in CloningVectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

A variety of expression systems exist for the production of thepolypeptides of the invention. EVs derived from fibroblast-like cellscan be loaded with any one or more of the following expression vectorsor with the polypeptides generated using such vectors. Expressionvectors useful for producing polypeptides include, without limitation,chromosomal, episomal, and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof.

One particular bacterial expression system for polypeptide production isthe E. coli pET expression system (e.g., pET-28) (Novagen, Inc.,Madison, Wis). According to this expression system, DNA encoding apolypeptide is inserted into a pET vector in an orientation designed toallow expression. Since the gene encoding such a polypeptide is underthe control of the T7 regulatory signals, expression of the polypeptideis achieved by inducing the expression of T7 RNA polymerase in the hostcell. This is typically achieved using host strains that express T7 RNApolymerase in response to IPTG induction. Once produced, recombinantpolypeptide is then isolated according to standard methods known in theart, for example, those described herein.

Another bacterial expression system for polypeptide production is thepGEX expression system (Pharmacia). This system employs a GST genefusion system that is designed for high-level expression of genes orgene fragments as fusion proteins with rapid purification and recoveryof functional gene products. The protein of interest is fused to thecarboxyl terminus of the glutathione S-transferase protein fromSchistosoma japonicum and is readily purified from bacterial lysates byaffinity chromatography using Glutathione Sepharose 4B. Proteins can berecovered under mild conditions by elution with glutathione. Cleavage ofthe glutathione S-transferase domain from the fusion protein isfacilitated by the presence of recognition sites for site-specificproteases upstream of this domain. For example, proteins expressed inpGEX-2T plasmids may be cleaved with thrombin; those expressed inpGEX-3X may be cleaved with factor Xa.

Alternatively, recombinant polypeptides of the invention are expressedin Pichia pastoris, a methylotrophic yeast. Pichia is capable ofmetabolizing methanol as the sole carbon source. The first step in themetabolism of methanol is the oxidation of methanol to formaldehyde bythe enzyme, alcohol oxidase. Expression of this enzyme, which is codedfor by the AOX1 gene is induced by methanol. The AOX1 promoter can beused for inducible polypeptide expression or the GAP promoter forconstitutive expression of a gene of interest.

Once the recombinant polypeptide of the invention is expressed, it isisolated, for example, using affinity chromatography. In one example, anantibody (e.g., produced as described herein) raised against apolypeptide may be attached to a column and used to isolate therecombinant polypeptide. Lysis and fractionation ofpolypeptide-harboring cells prior to affinity chromatography may beperformed by standard methods (see, e.g., Ausubel et al., supra).Alternatively, the polypeptide is isolated using a sequence tag, such asa hexahistidine tag (SEQ ID NO: 4), that binds to nickel column.

Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry and Molecular Biology,eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention,particularly short peptide fragments, can also be produced by chemicalsynthesis (e.g., by the methods described in Solid Phase PeptideSynthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Thesegeneral techniques of polypeptide expression and purification can alsobe used to produce and isolate useful peptide fragments or analogs(described herein).

The isolated polypeptides or fragments are loaded into EVs as describedherein.

Antibody Delivery

Like other polypepties, antibodies can be delivered using EVs derivedfrom fibroblast-like cells or CAFs. Antibodies can be made by any of themethods known in the art utilizing a polypeptide interest, orimmunogenic fragments thereof, as an immunogen. One method of obtainingantibodies is to immunize suitable host animals with an immunogen and tofollow standard procedures for polyclonal or monoclonal antibodyproduction. The immunogen will facilitate presentation of the immunogenon the cell surface. Immunization of a suitable host can be carried outin a number of ways. Nucleic acid sequences encoding a polypeptide ofthe invention or immunogenic fragments thereof, can be provided to thehost in a delivery vehicle that is taken up by immune cells of the host.The cells will in turn express the receptor on the cell surfacegenerating an immunogenic response in the host. Alternatively, nucleicacid sequences encoding the polypeptide, or immunogenic fragmentsthereof, can be expressed in cells in vitro, followed by isolation ofthe polypeptide and administration of the polypeptide to a suitable hostin which antibodies are raised.

Alternatively, antibodies against the polypeptide may, if desired, bederived from an antibody phage display library. A bacteriophage iscapable of infecting and reproducing within bacteria, which can beengineered, when combined with human antibody genes, to display humanantibody proteins. Phage display is the process by which the phage ismade to ‘display’ the human antibody proteins on its surface. Genes fromthe human antibody gene libraries are inserted into a population ofphage. Each phage carries the genes for a different antibody and thusdisplays a different antibody on its surface.

Antibodies made by any method known in the art can then be purified fromthe host. Antibody purification methods may include salt precipitation(for example, with ammonium sulfate), ion exchange chromatography (forexample, on a cationic or anionic exchange column run at neutral pH andeluted with step gradients of increasing ionic strength), gel filtrationchromatography (including gel filtration HPLC), and chromatography onaffinity resins such as protein A, protein G, hydroxyapatite, andanti-immunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineeredto express the antibody. Methods of making hybridomas are well known inthe art. The hybridoma cells can be cultured in a suitable medium, andspent medium can be used as an antibody source. Polynucleotides encodingthe antibody of interest can in turn be obtained from the hybridoma thatproduces the antibody, and then the antibody may be producedsynthetically or recombinantly from these DNA sequences. For theproduction of large amounts of antibody, it is generally more convenientto obtain an ascites fluid. The method of raising ascites generallycomprises injecting hybridoma cells into an immunologically naivehistocompatible or immunotolerant mammal, especially a mouse. The mammalmay be primed for ascites production by prior administration of asuitable composition (e.g., Pristane).

In particular embodiments, the EV comprises an antibody against a tumorantigen (e.g., an antigen associated with breast cancer tumor,pancreatic tumor, glioblastoma, melanoma, lung cancer tumor, ovariancancer tumor). In another embodiment, the antibody comprises an antibodythat targets a protein expressed in the blood vessels supplying thetumor. In yet another embodiment, the antibody targets a protein thatfunctions in miRNA maturation, checkpoint blocking, or that is histonespecific.

Small Molecule Delivery

EVs derived from fibroblast-like cells are used to deliver therapeuticor imaging agents. In one embodiment, the invention provides an EVcomprising, for example, N-fluorescein phosphatidylethanolamine(N-F-PE), doxorubicin, or cisplatin. In other embodiments, an EVdescribed herein a conventional chemotherapeutic agent including, butnot limited to, alemtuzumab, altretamine, aminoglutethimide, amsacrine,anastrozole, azacitidine, bleomycin, bicalutamide, busulfan,capecitabine, carboplatin, carmustine, celecoxib, chlorambucil,2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide,cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel,doxorubicin, epirubicin, estramustine phosphate, etodolac, etoposide,exemestane, floxuridine, fludarabine, 5-fluorouracil, flutamide,formestane, gemcitabine, gentuzumab, goserelin, hexamethylmelamine,hydroxyurea, hypericin, ifosfamide, imatinib, interferon, irinotecan,letrozole, leuporelin, lomustine, mechlorethamine, melphalen,mercaptopurine, 6-mercaptopurine, methotrexate, mitomycin, mitotane,mitoxantrone, nilutamide, nocodazole, paclitaxel, pentostatin,procarbazine, raltitrexed, rituximab, rofecoxib, streptozocin,tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan,toremofine, trastuzumab, vinblastine, vincristine, vindesine, andvinorelbine.

In particular embodiments, the EV comprises sirolimus, evirolimus,lapatinib, or olaparib.

Delivery of Imaging Agents

EVs comprising a detectable agent are useful for imaging studies. Theinvention provides an EV comprising any one of the following exemplarysmall molecules useful in imaging: carbocyanine, indocarbocyanine,oxacarbocyanine, thüicarbocyanine and merocyanine, polymethine,coumarine, rhodamine, xanthene, fluorescein, borondipyrromethane(BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750,AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750,AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547,Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.

In other embodiments, the EV comprises a nanoparticle useful in imagingstudies. In one embodiment, nanoparticles are synthesized using abiodegradable shell known in the art. In one embodiment, a polymer, suchas poly (lactic-acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) isused. Such polymers are biocompatible and biodegradable, and are subjectto modifications that desirably increase the circulation lifetime of thenanoparticle. In one embodiment, nanoparticles are modified withpolyethylene glycol (PEG), which increases the half-life and stabilityof the particles in circulation (Gref et al., Science 263(5153):1600-1603, 1994).

Biocompatible polymers useful in the composition and methods of theinvention include, but are not limited to, polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetage phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulphate sodium salt,poly(methyl methacrylate), poly(ethylmethacrylate),poly(butylmethacrylate), poly(isobutylmethacrylate),poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), poly(vinyl acetate, poly vinyl chloride polystyrene,polyvinylpryrrolidone, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodeclmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecl acrylate) and combinations of any of these. Inone embodiment, the nanoparticles of the invention include PEG-PLGApolymers.

In response to the growing need for encapsulation materials, severaldifferent routes to producing hollow polymeric capsules are available.In one example, the shell is composed of dendrimers (Zhao, M., et al. J.Am. Chem. Soc. (1998) 120:4877). A dendrimer is an artificiallymanufactured or synthesized large molecule comprised of many smallerones linked together—built up from branched units called monomers.Technically, dendrimers are a unique class of a polymer, about the sizeof an average protein, with a compact, tree-like molecular structure,which provides a high degree of surface functionality and versatility.Their shape gives them vast amounts of surface area, making them usefulbuilding blocks and carrier molecules at the nanoscale and they come ina variety of forms, with different physical (including optical,electrical and chemical) properties. In other embodiments, the shellcomprises block copolymers (Thurmond, K. B., II, et al. J. Am. Chem.Soc. (1997) 119:6656; Macknight, W. J., et al., Acc. Chem. Res. (1998)31:781; Harada, A. and Kataoka, K. Science (1999), 283:65), vesicles(Hotz, J. and Meier, W. Langmuir (1998) 14:1031; Discher, B. M., et al.,Science (1999) 284:1143), hydrogels (Kataoka, K. et al. J. Am. Chem.Soc. (1998) 120:12694) and template-synthesized microtubules (Martin, C.R. and Parthasarathy, R. V. Adv. Mater. (1995) 7:487) that are capableof encapsuling a photosensitizer.

In another embodiment, EV of the invention comprises an isotopic labelfor positron or scintillation or SPECT imaging.

In another embodiment, an EV of the invention comprises a magneticnanoparticle that has a high magnetic moment to enhance the selectivityof the nanoparticle for detection. In another embodiment, a magneticnanoparticle includes a magnetic core and a biocompatible outer shell,in which the outer shell both protects the core from oxidation andenhances magnetic properties of the nanoparticle. The enhanced magneticproperties can include increased magnetization and reduced coercivity ofthe magnetic core, allowing for highly sensitive detection as well asdiminished non-specific aggregation of nanoparticles. By formingbiocompatible nanoparticles having enhanced magnetic properties,detection of specific target proteins and cells is provided. In oneembodiment, a nanoparticle core is formed from ferromagnetic materialsthat are crystalline, poly-crystalline, or amorphous in structure. Forexample, the nanoparticle core can include materials such as, but notlimited to, Fe, Co, Ni, FeOFe₂O₃, NiOFe₂O₃, CuOFe₂O₃, Fe₂O₃, MgOFe₂O₃,MnBi, MnSb, MnOFe₂O₃, Y3Fe₅Oi₂, CrO₂, MnAs, SmCo, FePt, or combinationsthereof.

In another embodiment, the outer shell of the magnetic nanoparticlepartially or entirely surrounds the nanoparticle core. In someimplementations, the shell is formed from a superparamagnetic materialthat is crystalline, poly-crystalline, or amorphous in structure. Insome cases, the material used to form the shell is biocompatible, i.e.,the shell material elicits little or no adverse biological/immuneresponse in a given organism and/or is nontoxic to cells and organs.Exemplary materials that can be used for the shell include, but are notlimited to, metal oxides, e.g., ferrite (Fe₃C″4), FeO, Fe203, CoFe₂04,NiFe₂04, ZnMnFe₂04, or combinations thereof.

Methods of making and delivering nanoparticles are known in the art anddescribed, for example, in the following US Patent Publications:20150258222, 20140303022, 20130309170, and 20130195767.

Extracellular Vesicle Isolation, Loading, and Targeting

EVs defined herein are generated as described herein below. In general,the EVs are released by cells (e.g., CAFs, fibroblast-like cells) intothe extracellular environment. In vivo, EVs are isolated from a varietyof biological fluids, including but not limited to, blood, plasma,serum, urine, stool, semen, cerebrospinal fluid, prostate fluid,lymphatic drainage, bile fluid, and pancreatic secretions. The EVs arethen separated using routine methods known in the art. In oneembodiment, EVs are isolated from the supernatants of cultured cellsusing differential ultracentrifugation. In another embodiment, EVs areseparated from nonmembranous particles, using their relatively lowbuoyant density (Raposo et al., 1996; Escola et al., 1998; van Niel etal., 2003; Wubbolts et al., 2003). Kits for such isolation arecommercially available, for example, from Qiagen, InVitrogen and SBI.

Methods for loading EVs with agent are known in the art and includelipofection, electroporation, as well as any standard transfectionmethod.

In one embodiment, the EVs comprising a polynucleotide or polypeptide orsmall molecule of interest are obtained by over-expressing thepolynucleotide or polypeptide or loading the cells with the smallmolecule in culture and subsequently isolating indirectly modified EVsfrom the cultured cells. In another embodiment, EVs comprising apolynucleotide or polypeptide or small molecule of interest aregenerated by loading previously purified EVs with the molecule(s) ofinterest into/onto the EVs by electroporation (polynucleotide orpolypeptide), covalent or non-covalent coupling to the EV surface(polynucleotide or polypeptide or small molecule) or simpleco-incubation (polynucleotide or polypeptide or small molecule).

In general, the physical properties of EVs of the invention aresufficient to target the EV to a cancer cell of interest (e.g., breastcancer tumor, pancreatic tumor, glioblastoma, melanoma, lung cancertumor, ovarian cancer tumor). Nevertheless, in particular embodiments,it may be useful to derivatize the EV with an antibody that selectivelybinds to a tumor antigen. Targeted EVs may be loaded with an agent thatis particularly effective against the targeted cancer cell. Exemplarytarget factors and agents are provided in Table 1 (below).

Target Clinical factor Expression cell Function Drug Mechanism TrialVEGF tumor cells CAFs, TAMs. Angiogenesis Bevacizumab NeutralizationVEGF Phase II Adsflt Interception of VEGF Preclinical IMC-1C11anti-VEGFR-2 antibody Phase I RPI.4610 anti-VEGFR-1 ribozyme Phase IITenascin-C CAFs, cancer cells cell adhesion 81C6 radioimmunotherapyPhase II ATN-RNA siRNA Phase I FAP CAFs, TECs, cancer cells Serineprotease PT-100 activity inhibitor Phase I Sibrotuzumab anti-FAPantibody Phase I Sc40-FasL induce apoptosis of FAP⁺ preclinical cellsRebimastat activity inhibitor Phase III CTGF CAFs, TECs, cancer cell,Growth factor FG-3019 anti-CTGF antibody preclinical neural DN-9693degrade mRNA preclinical MMPs CAFs, TECs, TAMs, metalloproteinasesMarimastat activity inhibitor Phase III cancer cells Tanomastat activityinhibitor Phase III Rebimastat activity inhibitor Phase III uPA CAFs,TAMs, cancer cells Serine protease PAI-2 activity inhibitor preclinicaluPA-UT1 activity inhibitor preclinical CA IX CAFs, cancer cells Carbonicanhydrase Rencarex WX-G250 induce ADCC Phase III

Pharmaceutical Compositions

The invention provides EVs for the delivery of therapeutic compositionsthat specifically deliver an agent (e.g., polynucleotide, polypeptide,or small molecule for the treatment of disease. In one embodiment, thepresent invention provides a pharmaceutical composition comprising an EVderived from a CAF or stromal cell. EVs of the invention may beadministered as part of a pharmaceutical composition. In general, EVsare provided in a physiologically balanced saline solution. The solutioncomprising the EVs is stored at room temperature for up to about 24hours, for longer than twenty four hours such solutions can be stored atabout four degrees Celsius for days, weeks, or months. EVs are frozenfor long term storage up to 10 years. The compositions should be sterileand contain a therapeutically effective amount of the EV in a unit ofweight or volume suitable for administration to a subject.

EVs of the invention may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer thecompounds to patients suffering from a disease (e.g., cancer).Administration may begin before the patient is symptomatic. Anyappropriate route of administration may be employed, for example,administration may be parenteral, intravenous, intraarterial,subcutaneous, intratumoral, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal,intracisternal, intraperitoneal, intranasal, aerosol, suppository, ororal administration. For example, therapeutic formulations may be in theform of liquid solutions or suspensions; for oral administration,formulations may be in the form of tablets or capsules; and forintranasal formulations, in the form of powders, nasal drops, oraerosols.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” Ed. A. R.Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for microRNA moleculesinclude ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes. Formulations for inhalationmay contain excipients, for example, lactose, or may be aqueoussolutions containing, for example, polyoxyethylene-9-lauryl ether,glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

The formulations can be administered to human patients intherapeutically effective amounts (e.g., amounts which prevent,eliminate, or reduce a pathological condition) to provide therapy for adisease or condition. The preferred dosage of an EV of the invention islikely to depend on such variables as the type and extent of thedisorder, the overall health status of the particular patient, theformulation of the compound excipients, and its route of administration.

With respect to a subject having a neoplastic disease or disorder, aneffective amount is sufficient to stabilize, slow, or reduce theproliferation of the neoplasm. Generally, doses of active polynucleotidecompositions of the present invention would be from about 0.01 mg/kg perday to about 1000 mg/kg per day. It is expected that doses ranging fromabout 50 to about 2000 mg/kg will be suitable. Lower doses will resultfrom certain forms of administration, such as intravenousadministration. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. Multiple dosesper day are contemplated to achieve appropriate systemic levels of EVs

A variety of administration routes are available. The methods of theinvention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. Other modes of administrationinclude oral, rectal, topical, intraocular, buccal, intravaginal,intracisternal, intracerebroventricular, intratracheal, nasal,transdermal, within/on implants, e.g., fibers such as collagen, osmoticpumps, or grafts comprising appropriately transformed cells, etc., orparenteral routes.

Therapy

Results provided herein below show that conditioned media from cancercells can be used to alter a fibroblast-like cell's gene expression andphysiology to promote cancer growth. These cancer-promoting changes infibroblast-like cells include changes in the exosomes/EVs (and othersignals) that they deliver to neighboring cancer cells. The inventionprovides methods for using a fibroblast-like cell-derived EVs to reversethese changes and inhibit cancer cell growth in vitro and in vivo. Inone embodiment of the present invention, fibroblast-like cell-derivedexosomes/EVs can be engineered to deliver anti-neoplastic, therapeuticmiRs in vivo. In this embodiment, the fibroblast-like cells representboth localized cells of endothelial origin, localized tissuepleuripotential stem cells which develop fibroblast phenotypes orendogenous stem cells of bone marrow origin which have migrated to thesite of tumor.

Yet another embodiment of the present invention is a cancer therapy thatinterrupts the support that stroma provides to cancer cells, in thecontext of CCA, in the context of breast cancer, and more broadly withpotential to all cancers. Although there are therapeutic strategies tokill cancer cells (from conventional chemotherapy to targeted moleculartherapies), there are currently no FDA-approved therapies to interruptthe support that stroma provides to cancer cells. Another embodiment ofthe present invention utilizes EV-mediated miR transfer from stromalcells to cancer cells to create a therapeutic with anti-neoplastic andsurvival-extending properties in vivo.

Other embodiments of the present invention target other cancers,including breast cancer, as well as cancers with pronounced fibrosis.Some of the most aggressive cancers, such as pancreatic, breast, andhepatocellular carcinoma, develop a close symbiotic relationship withfibroblast-like cells, and we have shown that this relationship hasstrong supporting effects on both CCA and breast cancer cells

Therapy may be provided wherever cancer or other disease therapy isperformed: at home, the doctor's office, a clinic, a hospital'soutpatient department, or a hospital. Treatment generally begins at ahospital so that the doctor can observe the therapy's effects closelyand make any adjustments that are needed. The duration of the therapydepends on the kind of cancer being treated, the age and condition ofthe patient, the stage and type of the patient's disease, and how thepatient's body responds to the treatment. Drug administration may beperformed at different intervals (e.g., daily, weekly, or monthly).Therapy may be given in on-and-off cycles that include rest periods sothat the patient's body has a chance to build healthy new cells andregain its strength.

Depending on the type of disease and its stage of development, thetherapy can be used to slow the spreading of the cancer, to slow thecancer's growth, to kill or arrest cancer cells that may have spread toother parts of the body from the original tumor, to relieve symptomscaused by the cancer, or to prevent cancer in the first place. Asdescribed above, if desired, treatment with an agent of the inventionmay be combined with conventional therapies, including therapies for thetreatment of proliferative disease (e.g., radiotherapy, surgery, orchemotherapy). For any of the methods of application described above, anEV of the invention is desirably administered intravenously or isapplied to the site of neoplasia (e.g., by injection).

In particular embodiments, EVs can be used to deliver therapeutic miRsin vivo, without obvious involvement of normal liver cells nordevelopment of a cellular inflammatory reaction. Furthermore, thespecific finding is that CAF derived EV-based therapy utilizing miRsdelivered by EVs in particular embodiments herein targets thecancer-stroma niche interactions, an important property of cancers thatis not currently addressed by prior art nor any FDA-approved agents. EVscontribute to CAF-mediated support of CCA, and that miR-loaded,fibroblast-derived EVs can slow the growth of CCA and prolong survivalin vivo. One embodiment of the present invention is a therapeutic withanti-proliferation, anti-spread and with survival-extending propertiesin vivo.

Kits

Kits of the invention include EVs comprising an agent formulated fordelivery to a cell in vitro or in vivo. Optionally, the kit includesdirections for delivering the EV to a subject. In other embodiments, thekit comprises a sterile container which contains the EV; such containerscan be boxes, ampules, bottles, vials, tubes, bags, pouches,blister-packs, or other suitable container form known in the art. Suchcontainers can be made of plastic, glass, laminated paper, metal foil,or other materials suitable for holding nucleic acids. The instructionswill generally include information about the use of the EV. In otherembodiments, the instructions include at least one of the following:description of the EV; methods for using the enclosed materials for thetreatment of a disease, including a cancer; precautions; warnings;indications; clinical or research studies; and/or references. Theinstructions may be printed directly on the container (when present), oras a label applied to the container, or as a separate sheet, pamphlet,card, or folder supplied in or with the container.

Trangenic Animals

In another aspect, the EVs of the present invention can be used tocreate animal models (including large animals such as swine, canine,primate and the like) of particular diseases including, but not limitedto, cancer. For example, the EVs can be manipulated to contain geneticmaterial comprising a transposon system (e.g., sleeping beauty) encodingan oncogene. In another embodiment, genetic material comprises a plasmidencoding an oncogene. In a further embodiment, the genetic materialcomprises a viral vector encoding an oncogene. The oncogene can include,but is not limited to, one or more of c-Myc, K-Ras, N-Ras, c-Met, AKT,P53, P16, CTNNB1, AXIN1, AXIN2, TP53, PIK3CA, PTEN, MET.

Another embodiment of the present invention utilizes one or more of thetherapies described in the present patent application in conjunctionwith one or more cancer therapies, such as surgery, chemotherapy,radiation, and targeted molecular therapies.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention tothe fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods described andclaimed herein are made and evaluated, and are intended to be purelyillustrative and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for herein. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees Celsius or is at ambient temperature, and pressure is at or nearatmospheric. There are numerous variations and combinations of reactionconditions, e.g., component concentrations, desired solvents, solventmixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Cholangiocarcinoma (CCA), pancreatic cancer, and other cancers induce astrong desmoplastic reaction that includes intimate contact betweencancer cells and tissue fibroblasts.

The vast majority of hepatocellular cancers (HCC) arise in a fibroticliver. This fibrotic response was long assumed to have antineoplasticeffects, but recent studies support a new paradigm in whichcancer-associated fibroblasts (CAFs) play a supporting role in cancergrowth and metastasis (Mueller M M, Fusenig N E. Friends or foes—bipolareffects of the tumor stroma in cancer. Nat Rev Cancer 2004; 4:839-49).For example, removing CAFs inhibits CCA growth, while CAF-derived PDGF,Periostin, Tenascin-C, Thrombospondin-I, and Galectin-1 are known topromote tumor growth (Sirica A E, Dumur C I, Campbell D J, et al.Intrahepatic cholangiocarcinoma progression: prognostic factors andbasic mechanisms. Clin Gastroenterol Hepatol 2009; 7:S68-78; Kawahara N,Ono M, Taguchi K, et al Enhanced expression of thrombospondin-1 andhypovascularity in human cholangiocarcinoma. Hepatology 1998; 28:1512-7;Shimonishi T, Miyazaki K, Kono N, et al. Expression of endogenousgalectin-1 and galectin-3 in intrahepatic cholangiocarcinoma. Hum Pathol2001; 32:302-10). These observations fit within a broader paradigm inwhich cancer cells prime stroma to support cancer growth and metastasis(Wan L, Pantel K, Kang Y. Tumor metastasis: moving new biologicalinsights into the clinic. Nat Med 2013; 19:1450-64; Hood J L, San R S,Wickline S A. Exosomes released by melanoma cells prepare sentinel lymphnodes for tumor metastasis. Cancer Res 2011; 71:3792-801) in abidirectional interplay of signaling reactions (Roccaro A M, Sacco A,Maiso P, et al. BM mesenchymal stromal cell-derived exosomes facilitatemultiple myeloma progression. J Clin Invest 2013; 123:1542-55).

Example 1: Co-Culturing Fibroblasts and Cancer Cells Leads toDown-Regulation of Specific miRs within Fibroblasts

The hypothesis that cancer cells derive support from their stroma isbased on a number of observations, one of which is the fact that cancercells alter the physiology and gene expression patterns of theirsurrounding cells. In the case of CCA and other gastrointestinal tumors,tissue fibroblasts and their extracellular matrix (ECM) are majorcomponents of the tumor stroma. To determine if CCA cancer cells affectfibroblast gene expression patterns, we generated fluorescent CCA celllines by infecting them with MSCV-IRES-EGFP (MIEG3), a retrovirus thatexpresses enhanced-GFP (EGFP) (Olaru A V, Ghiaur G, Yamanaka S, et al. AmicroRNA downregulated in human cholangiocarcinoma controls cell cyclethrough multiple targets involved in the G1/S checkpoint. Hepatology2011). These cells were then co-cultured with LX2 cells (FIG. 1) for 14days to mimic in vitro the close interactions between CAFs and cancercells that occur in vivo. The LX2 cells were then separated from thefluorescent cancer cells by FACS, lysed, followed by RNA extraction andqRT-PCR analysis to identify changes in fibroblast miR abundance inducedby CCA cancer cells.

We identified significant decreases in multiple fibroblast miRs,including miR-195, miR-192 and miR-126 (FIG. 2). These miRs representcandidate genes involved in the transition from normal fibroblasts toCAFs. We initially focused on the possible role of miR-195 in thisprocess, since earlier studies of fibroblast activation had demonstratedthat miR-195 is repressed during the differentiation of quiescentfibroblasts to activated, collagen-producing fibroblasts (Maubach G, LimM C, Chen J, et al. miR studies in in vitro and in vivo activatedhepatic stellate cells. World J Gastroenterol; 17:2748-73; Lakner A M,Steuerwald N M, Walling T L, et al. Inhibitory effects of microRNA 19bin hepatic stellate cell-mediated fibrogenesis. Hepatology; 56:300-10;Chen C, Wu C Q, Zhang Z Q, et al. Loss of expression of miR-335 isimplicated in hepatic stellate cell migration and activation. Exp. CellRes.; 317:1714-25).

Example 2: Up-Regulation of miR-195 within Fibroblasts is Sufficient toInhibit Cancer Cell Invasiveness

In vitro, CCA cells display significantly higher invasion in a matrigelassay when they are co-cultured with LX2 cells. To determine whetherrestoring miR-195 expression in LX2 cells had any effect on theinvasiveness of co-cultured CCA cells, we generated an LX2 fibroblastcell line (LX2-195) that had restored expression of miR-195, and askedif this had any effect on the behavior of CCA cells in co-culture.Specifically, we co-cultured LX2-195 cells and control LX2 cells(expressing a non-specific inhibitor miR mimic (NSM)) with fourdifferent cancer cell lines: HuCCT1, SG231, BDENeu, and BDESp, all ofwhich are intrahepatic CCA cell lines, and then assayed them for cancercell invasiveness by staining the matrigel with Crystal violet andcounting the number of invading cells (neither LX2 control nor LX2-195cells invade the matrigel). Co-culturing cancer cells with LX2-195 cellsresulted in a significant reduction in the invasiveness of all fourcancer cell lines examined (FIG. 3). The most parsimoniousinterpretation of these results is that reversing the reduction of asingle miR species in fibroblasts, miR-195, was sufficient to inhibitthe invasion of co-cultured cancer cells.

Example 3: LX2-195 Cells Release a Diffusible Factor that InhibitsCancer Cell Invasion, Migration, and Growth

The inhibition of cancer cell invasiveness by co-culture with LX2-195cells (demonstrated above) could be mediated by either direct cell-cellcontact and/or by diffusible factors released from LX2-195 cells. Giventhat diffusible factors have the potential for future therapeuticdevelopment, we tested whether LX2-195 cells impacted cancer cellphenotypes in the absence of direct cell-cell contact. In brief, cancercells and fibroblasts were grown on opposite sides of a transwellapparatus with ˜400 nm dia. pores for a period of 5 days (FIG. 4). Thecancer cells were then removed and assayed for invasiveness, migration,and growth. Cancer cells exposed to diffusible factors released fromLX2-195 cells displayed significant reductions in invasion, migration,and growth, as compared to cancer cells that had been co-cultured withcontrol LX2 cells, which have much lower levels of miR-195.

Example 4: LX2-195 Cells Release a Diffusible Factor that CausesUp-Regulation of miR-195 in Neighboring Cancer Cells

We next tested whether the soluble factors released by LX2-195 cellsaffected the levels of miR-195 in neighboring cancer cells. Using thetranswell assay, cancer cells were exposed to conditioned media fromcontrol LX2 or LX2-195 cells. Cancer cells were then purified away fromthe LX2 cells by FACS, and RNA was extracted from the cancer cells andprocessed for qRT-PCR to determine the levels of miR-195 and controls.We found that the level of miR-195 in cancer cells was significantlyup-regulated following exposure to diffusible factors released byLX2-195 cells (FIG. 5).

Example 5: Fibroblast-Like Cells Secrete miR-195 in ExtracellularVesicles (EVs)

To explore the possibility that EVs might contribute to the CAF-cancerinteractions outlined in the previous experiments, we asked whetherfibroblast-like cells release miR-195 in EVs, and whether the levels ofvesicle-associated miR-195 were higher in vesicles released by LX2-195cells. EVs were collected from the supernatant of control LX2 cells andof LX2-195 cell cultures, followed by RNA extraction and qRT-PCR todetermine the relative abundance of miR-195 in the two EV preparations.We observed that control LX2 cells (expressing a non-specific mimic(NSM)) secrete EVs that contain detectable levels of miR-195. However,the levels of miR-195 were >60-fold higher in the in EVs produced byLX2-195 cells (FIG. 6).

Example 6: Fibroblast-Like Cell-Derived Extracellular Vesicles (EVs) areSelectively Targeted to Tumor Cells In Vivo

The possibility that EVs might contribute to the CAF-cancer signalingobserved above, led us to ask whether fibroblast-like cell-derived EVsmight be targeted to tumor cells in vivo. To this end, we generated LX2cells that constitutively express TSG101/mCherry fusion protein (TSG101is secreted from the cell in EVs (Raposo G, Stoorvogel W. Extracellularvesicles: exosomes, microvesicles, and friends. J Cell Biol 2013;200:373-83), which allowed us to selectively detect thesefibroblasts-derived EVs). EVs from the resulting cell line,LX2-TSG101/mCherry, were collected from the supernatant by standardprocedures. The purified, TSG101/mCherry-labeled, fibroblast-derived EVswere then injected into the tail vein of rats, which had been injectedwith BDEneu tumor cells 24 days earlier and thus had already developedCCA in their liver (we have extensive experience with this model of CCA;see Sirica A E, Zhang Z, Lai G H, et al. A novel “patient-like” model ofcholangiocarcinoma progression based on bile duct inoculation oftumorigenic rat cholangiocyte cell lines. Hepatology 2008; 47:1178-90).24 hours after injection, we sacrificed the rats, removed their livers,lungs, and kidneys, generated slides of these tissues, and processedthem for immunofluorescence microscopy using antibodies specific foralpha-Smooth Muscle Actin (stains activated, collagen-producingfibroblasts) and mCherry to detect the fibroblast-derived,TSG101-mCherry-containing exosomes/EVs. These experiments revealed thatthe fibroblast-derived vesicles were highly enriched in “pockets” ofcancer cells within the fibrotic CCA mass in the liver (FIG. 7). We wereunable to detect significant staining for TSG101/mCherry innon-cancerous areas of the liver, the lung, or the kidney. Theseexperiments can be carried out using EGFP-containing EVs andtdTomato-expressing cancer cells, and the tissue sections are processedby immunogold label electron microscopy.

Example 7: Fibroblast-Like Cell-Derived Extracellular Vesicles (EVs) canSelectively Deliver Heterologous Proteins to Tumor Cells In Vivo

EVs from the cell line, LX2-TSG101/mCherry, were collected from thesupernatant by standard procedures. The purified,TSG101/mCherry-containing, fibroblast-derived EVs were then injectedinto the tail vein of rats, which had been injected with BDEneu tumorcells 24 days earlier and thus had already developed CCA in their liver(we have extensive experience with this model of CCA; see Sirica A E,Zhang Z, Lai G H, et al. A novel “patient-like” model ofcholangiocarcinoma progression based on bile duct inoculation oftumorigenic rat cholangiocyte cell lines. Hepatology 2008; 47:1178-90).24 hours after injection, we sacrificed the rats, removed their livers,lungs, and kidneys, generated slides of these tissues, and processedthem for immunofluorescence microscopy using antibodies specific foralpha-Smooth Muscle Actin (stains activated, collagen-producingfibroblasts) and mCherry to detect the fibroblast-derived,TSG101-mCherry-containing exosomes/EVs. These experiments revealed thatthe fibroblast-derived vesicles were highly enriched in “pockets” ofcancer cells within the fibrotic CCA mass in the liver (FIG. 7). We wereunable to detect significant staining for TSG101/mCherry innon-cancerous areas of the liver, the lung, or the kidney (data notshown). This point is also established by our demonstration that humanmammary fibroblast-derived EVs containing an exosomal form of GFP weretaken up by human breast cancer cells (FIGS. 16A-16D).

Example 8: Fibroblast-Like Cell-Derived Extracellular Vesicles (EVs) canSelectively Deliver DNA to Tumor Cells In Vivo

To further investigate the ability of EVs to selectively deliver cargomolecules to cancer cells, we generated BDEneu cells carrying aCre-reporter gene, CAG-loxP-tdTomato-loxp-EGFP. These cells displaybright red fluorescence due to the expression of tdTomato, (seen aswhite or light signal emitting cells on black and white drawings usingrespective optical filters) as but switch from red to green fluorescencefollowing the expression of Cre, which removes the tdTomato gene andplaces the promoter proximal to the EGFP gene (seen as white or lightsignal emitting cells on black and white drawings using respectiveoptical filters). These cells were injected into rats, and after tumorswere established the rats were treated with a single set of tail veininjections with EVs that had been loaded with a plasmid DNA designed toexpress Cre recombinase in mammalian cells. As shown in FIG. 8, asignificant number of the Cre-reporter CCA cells switched from red togreen fluorescence, demonstrating that fibroblast-like cell-derived EVscan deliver DNA to CCA cells in vivo. The fact that some cancer cellsretained their original red fluorescence is an outstanding control thatpoints to the efficacy of this assay system for optimizing the variablesin the experiment, as well as an internal control to ensure that thecorrect cells were used. Animals that were not injected withplasmid-loaded EVs failed to produce any green CCA cells. optimize ourtherapeutic technology.

Example 9: Fibroblast-Derived, miR-195 Mimic-Loaded EVs Inhibit CCAGrowth In Vivo in a Rat Model

Taken together, these observations indicate that fibroblast-derived EVscould be used to deliver therapeutic miRs to CCA in vivo. To explorethis hypothesis, we collected EVs from the supernatant of LX2 cells,loaded them with a miR-195 mimic by transfection using LipofectamineRNAiMAX (Invitrogen), and re-purified the loaded EVs by size exclusionchromatography. We also generated control EVs, by transfecting EVs fromsame LX2 cells with a non-specific miR mimic (NSM). qRT-PCR analysisindicated that miR-195 transfection of vesicles resulted in levels ofmiR-195 that were 500,000 times higher than in the control EVs. Todetermine whether these miR-195-loaded EVs might inhibit CCA growth invivo, we injected six rats with BDEneu cells, as previously described(Sirica A E, Zhang Z, Lai G H, et al. A novel “patient-like” model ofcholangiocarcinoma progression based on bile duct inoculation oftumorigenic rat cholangiocyte cell lines. Hepatology 2008; 47:1178-90).5 days later, the rats were injected (via tail vein) with either miR-195mimic-loaded or control miR-loaded EVs (at equivalent numbers of EVs andequivalent dose of miR mimic). Injections were repeated every other dayuntil day 35, at which time the animals were sacrificed and livers wereexcised. Morphological examination revealed that the tumor size wasreduced in all three animals that had been injected with the miR-195mimic-loaded EVs, relative to the three animals that had been injectedwith the NSM-loaded EVs (FIGS. 9 and 10). Based on our experience withthis animal model, tumor growth in the control-treated animals wassimilar to what occurs in untreated animals (see Sirica A E, Zhang Z,Lai G H, et al. A novel “patient-like” model of cholangiocarcinomaprogression based on bile duct inoculation of tumorigenic ratcholangiocyte cell lines. Hepatology 2008; 47:1178-90).

Example 10: Treatment with miR-195 Loaded EVs Downregulates CDK6 andVEGF (Known Targets of miR-195) in Cancer Cells

To test whether miR-195 causes expected changes in gene expression, wemeasured CDK6 and VEGF mRNA, two reported miR-195 targets (FIG. 11).Both mRNAs were downregulated in all experimental paradigms, including(left panel) direct transfection of BDEneu cells, (middle panel)exposure of cells to conditioned medium of LX2-miR-195, and (rightpanel) cells incubated with miR-195-loaded EVs.

Example 11: Treatment of Rats with CCA via Tail Vein with EVs-miR-195Increases their Survival Significantly

We treated 20 cancer-bearing rats with EVs-195 and with EVs-NSM(control), respectively. The treatment was commenced post cancer celltransplantation Day 15 (as to not interfere with the implantation oftumor cells, nor with the early steps of cancer development and growth).Treatment was continued till rats died due to cancer. All experimentshad been approved by the Hopkins IACUC. As shown in FIG. 12, ratstreated with EVs-195 displayed a statistically significant, 50% increasein survival, providing solid evidence that miR-loaded EVs can have apositive therapeutic effect in vivo.

Example 12: To Identify the Optimal Parameters of miR-195 Loaded EVTherapy in a Rat CCA Model

The purpose of the in vivo experiments was to assess if intravenoustreatment with EVs loaded with a miR species works in treating CCA. Asdescribed herein, we will test several conditions with the purpose ofelucidating the rate-limiting factors playing a role in the efficiencyof this treatment.

Frequency of treatment: In the preliminary experiments, we have treatedrats via tail vein every other day. We now propose to assess if lessfrequent treatment works equally well. We will do this study on 18 rats,as follows: 6 rats will be the control group (treated as in ourpreliminary experiments every other day), 6 rats will be treated every 4days and 6 rats will be treated every 7 days. After 30 days oftreatment, all rats will be euthanized and tumors measured.

Timing of treatment: In our preliminary experiments we started treatingrats 5 days after BDEneu cancer cell implantation in rat livers.Although not very likely, it is possible that miR-195 delivered by EVsaffected tumor implantation in addition to tumor growth. To elucidatethis aspect, we will now allow the tumors to develop for 2 weeks, thenthe treatment with miR-195-EVs will commence. We chose 2 weeks because,from our previous experiments, we know that these rats have tumorsdeveloped already by week 2, and some of them die of cancer at week 4.We will compare the treatment efficiency with the control arm from theexperiment above. For this experiment we will require 6 rats.

miR dose and EV dose: For preliminary experiments, for each rat, weutilized 200 μg miR mimic to transfect 200 μg of EVs (based on proteinweight, ratio 1:1) before delivering in vivo. However, we would like totest if a different miR dose or different EV dose is more efficient intreating CCA or, if the efficiency is maintained while decreasing thedose (with the purpose of cost saving). First, we will perform an invitro experiment to determine the best miR to EV ratio. We propose toutilize miR to transfect EVs in a weight ratio of 0.5:1, 1:1, 2:2 and4:1. Next, we will utilize these transfected EVs to treat HuCCT1 cellsin vitro and then determine by qRT-PCR the level of miR upregulation asa function of miR quantity used to transfect EVs. We utilize aconcentration of miR mimic of 15 μg per μL. We measure the amount ofexosomes based on the weight of the protein content. We usually extractapproximately 30 μg of exosomes from one 150 cm cell culture dish. Next,we will utilize the weight ratio miR:EVs that was determined in vitrofor all following experiments. We will then vary the amount of miRdelivered per rat (with the associated quantity of EVs). We will have 4experimental arms: 50 μg of miR mimic per injection/rat, 100 μg, 200 μgand 400 μg. Each experimental arm will include 6 rats for a total of 24rats. We will keep all other parameters of the experiments constant asin our preliminary experiments presented above (injection timing-5 dayspost-cancer implantation and injections every other day).

Kaplan-Meyer curves/survival: Once the optimal frequency, timing anddose of the treatment is established, we will perform an experiment todetermine the survival of rats treated with EV-miR-195. The control armwill include 12 rats treated with EV-NSM (negative control, EVstransfected with control miR) and the treatment arm will include 12 ratstreated with EV-miR195. We will record the date of death and thereforebe able to perform Kaplan-Meyer curves. These experiments will offervaluable information from a clinical perspective, as the size of thetumor is important, however, survival is also of utmost importance.While we have already demonstrated that EV injections can prolongsurvival (FIG. 11), survival studies will remain a key end-point as westrive to develop an effective anti-cancer therapy based on miR-loadedEVs.

Example 13: To Characterize the CCA Phenotype Induced by EVs Loaded withmiR-126, and -192, Respectively

miR-126 and -192 were among the top 3 candidate miRs in our screen. Infact, miR-126 and -192 were more strongly depleted in response to CCAcells than miR-195. We will pursue the same sets of experiments onmiR-126 and miR-192 that we have performed and proposed for miR-195. Forexample, we will generate LX2 cells that express miR-126, and cells thatexpress miR-192, and compare the effect that these cells have on theneoplastic properties of co-cultured CCA cells, relative to control LX2cells expressing a non-specific mimic (NSM)), both in direct co-cultureassays and in transwell co-culture assays. In fact, we have alreadygenerated a LX2-126 line, and our initial experiments indicates thatLX2-126 cells inhibit CCA invasiveness (FIG. 13). FIG. 13 shows LX2cells expressing miR-126 inhibit CCA invasiveness in vitro. HuCCT1 cellswere co-cultured directly with LX2 cells expressing either (upper image)a control miR, or (lower image) miR-126. Invasiveness of HuCCT1 cellswas decreased 3.2 fold when co-cultured with LX2-126 cells.

In addition, the LX2-126 cells appeared to inhibit CCA migration in ascratch assay test (FIG. 14). FIG. 14 shows LX2 cells expressing miR-126inhibit CCA migration 4-fold in vitro. HuCCT1 cells were co-cultureddirectly with LX2 cells expressing either a controls miR or miR-126.Migration was measured in a scratch assay.

As we move forward with these studies, from cells to miR-loaded EVs, andfrom in vitro experiments to in vivo experiments, we will alsoincorporate similar controls as those outlined previously, includingcharacterization of miR-loaded EVs (by immunoEm, differentialcentrifugation & immunoblot, etc.).

As for the in vivo experiments, they will mirror those shown andproposed for miR-195-loaded EVs. Specifically, we will inject miR-loadedEVs into the tail vein of rats that were previously induced to developiCCA. We will next determine differences in size of tumors in thetreatment vs. control arm. We will also perform experiments to determinethe optimal dose of EVs, miR loaded into EVs, the duration of treatment,frequency of injections and finally derive Kaplan-Meyer curves toindicate if there is a change in survival of rats treated with EV-miRsby tail vein. To determine the molecular & cellular impact of treatmentwith EV-loaded miR-126 and -192, we will perform experiments as outlinedunder Aim 1a. In brief, we will collect CCA cells (in vivo experimentsand in vitro culture), extract RNA, measure the levels of known miR-126and miR-192 targets, perform RNA-seq, followed by pathway analyses, andfollow-on experiments to identify the mechanisms by which injected EVsinhibit cancer growth (if they do).

Example 14: Delivery of a Small Molecule to Cancer Cells

MDA-MB-231 breast cancer cells (red) were incubated with EVs obtainedfrom human mammary fibroblast cells that had been incubated previouslywith the exosomal lipid N-F-PE. These EVs were taken up by the breastcancer cells, demonstrating that fibroblast-like cell-derived EVs can beloaded with small molecules and selectively deliver the small moleculesto cancer cells.

The results reported herein above were carried out using the followingmethods and materials.

Cell Culture

LX2 is a human liver stellate cell line derived to study fibrogenesis.HuCCT1 was derived from a patient with a moderately differentiatedadenocarcinoma of the intrahepatic biliary tree HuCCT1 was establishedfrom a patient with moderately differentiated adenocarcinoma of theintrahepatic biliary tree. SG231 is a cholangiocarcinoma cell linederived as described 41. TFK1 is an extrahepatic CCA cell line. BDENeuand BDEsp are rat intrahepatic CCA cell lines derived as described. RGFis a rat portal fibroblast cell line established by Fausther et al 21,22. H69, a gift from Dr. Jefferson (Tufts University, Boston, Mass.),are normal human intrahepatic cholangiocytes derived from a normal liverprior to liver transplantation. All cell lines were maintained and grownas described previously.

EVs Preparation and Characterization

EVs were separated via ultracentrifugation as described before from LX2cell culture medium that had been cultured for 72 hours with EV freeFBS. Multi-parameter nanoparticle optical analysis (Nanosight) andTransmission Electron Microscope (TEM) were utilized to determine theshape, size and tracking the brownian movement of EVs. Western blot forEV-specific proteins was performed with anti-CD63 antibody (Santa CruzDallas, Tex.) and anti-TSG-101 antibody (Abcam Cambridge, Mass.) asdescribed before.

miRNA Mimic Loading of EVs

Lipofectamine RNAiMAX (Life Technologies, NY) was used to transfect miRmimic into EVs with an adjusted protocol according to manufacturer'sinstruction. MiR-195 mimic and NSM were purchased from Dharmacon GEHealthcare. Then free miR-195 mimics were isolated with a micropartitionsystem (Vivaspin 2, 50 kDa MWCO PES, GE Healthcare, Laurel, Md.). Themixed suspension was added into the filter and centrifuged at 1500 g for5 min, the supernatant collected and either placed on the top of cellsfor in vitro experiments or used to inject into the CCA rat model in thein vivo experiments.

RNA Extraction

RNA from EVs was extracted by a modified Trizol method while spiking incel-miR-39 during the lysis step. Cells were lysed with Trizol reagent(Invitrogen, Carlsbad, Calif.) according to the manufacture's protocol.

MicroRNA Real Time PCR Array

LX2 and HuCCT1 MIEG3-EGFP were seeded at same amounts in flasks, thendirectly co-cultured for 14 days, EGFP-negative cells were sorted andcollected by FACS, followed by RNA extraction. 100 m RNA were used forRT-PCR array for co-cultured LX2 cells with LX2 cultured alone ascontrol. Following analysis, select miRs that were down-regulated inco-cultured LX2 cells were selected for follow-up experiments.

Quantitative RT-PCR (qRT-PCR) for miRs Expression

qRT-PCR was performed to detect the miR-195 expression in EVs, CCA celllines, and CCA tumor mass cells. For miR-195 expression in EVs, we usedcel-miR-39 as control, while for miR-195 expression in cells, RNU6B wasused to normalize the data as described before.

qRT-PCR for mRNA Expression

RNA was reversed transcribed according to the manufacture protocol(Thermo Scientific), IQ SYBR Green Supermix (Bio-Rad, Hercules, Calif.)was for used real time PCR amplification, GAPDH was used to normalizemRNA expression level, and melting curve analysis was used to confirmthe PCR results.

Plasmids, Transfection and Lentivirus/Retrovirus Infection

Vectors were purchased from Addgene (Cambridge, Mass.). Viralsupernatants were produced by transfection of HEK-293T cells with apackaging plasmid (pVSV-G). BDEneu cells were infected with viralsupernatant with Polybrene at a final concentration of 8 μg/μl.pCDH1-EF1-mCherry-TSG101-IRES-GFP was used to infecft LX2 cells. GFPpositive cells were sorted and used to isolate EVs with mCherry on thesurface. pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE was used as above toproduce viral particles, that were then used to infect BDEneu cells.Puromycin was used to select for stably infected cells. When aCre-recombinase encoding plasmid is detected, cells switch color fromRFP to GFP after excision of the loxp-dsRed-loxP element.

Conditioned Media Preparation

Confluent LX2-miR-195 mimic/NSM cells were incubated with DMEMsupplemented with penicillin/streptomycin, 0% FBS. Three days later, theconditioned media was collected, filtered, and used immediately.

Cell Invasion/Migration Assay

Cell invasion assays were performed using invasion chambers (Cat#354480, Corning, Tewksbury, Mass.) while for cell migration assays 8 μmTranswell plates (Cat #3422, Corning, Tewksbury, Mass.) were used. Forboth assays, DMEM with 10% FBS was placed in the lower chamber aschemoattractant. For directed transfection of CCA lines, 2 days aftertransfection, 3×10⁴ were seed into invasion chamber. For co-cultureinvasion assay, either 3×10⁴ HuCCT1, BDEneu, SG231 or BDEsp wereco-cultured with 3×10⁴ LX2-NSM or LX2-miR-195 mimc cells orRGF-NSM/miR-195 mimic at 37° C. for 2 days, then 6×10⁴ cells werediluted in serum-free medium and placed into the upper chambers. After48 hours the non-invading/non-migrating cells were removed from themembrane upper surface with cotton swabs, and invaded/migrated cells onthe lower side of chamber were stained with crystal violet. Cells werecounted in 3 random fields at a magnification of 200×.

Proliferation of CCA Cell Lines Co-Cultured with LX2/RGF miR-195Mimic/NSM, and CCA Cell Lines Directly Transfected with miR-195Mimic/NSM

LX2 or RGF cells transfected with miR-195 mimic or NSM were cultured for2 days, then washed and trypsinised, centrifuged and washed 3 times withPBS. After cell counting, 5×10⁴ LX2 or RGF cells were seeded into theupper well of 12 well 0.4 μm transwell co-culture system (cat #3460,Corning, Tewksbury, Mass.). For the bottom chamber, 1×10⁵ CCA cells(BDEneu, HuCCT1) were seeded at the same time after trypinization andcounting as above. After 5 days, the CCA cell number was determined bycounting. For directly transfected BDEneu and HuCCT1 with miR-195 mimicor NSM, 1000 cells were seeded into 96 well plates, MTs Assay (CellTiter96 Aqueous One solution Cell Proliferation Assay Cat # G3580, PromegaCorporation, Sunnyvale, Calif.) was used to determine the proliferation.

Animal Studies

Fischer 344 male (150-170 g) were purchased from Harlan (Frederick, Md.)and housed in the animal facility at Johns Hopkins University. Allanimal work was approved by and conducted in accordance with theguidelines of the Institutional Animal Care and Use Committee at theJohns Hopkins University. For the CCA rat model Fischer 344 male ratswere anesthetized and then inoculated with 1×10⁶ BDEneu cells in 100 μlHBSS injected into the left liver lobe, followed by ligation of thecommon bile duct. Rats were monitored daily until day 5 or day 15, whenthe treatment with miR-loaded EVs started.

Treatment of CCA Rats with miR-195-Loaded EVs

Initially 6 CCA rats were randomized into 2 groups. Starting at day 5,the rats received EV loaded with miR-195 mimic/NSM via tail veilinjections every 2 days. After 25 days, rats were sacrificed, tumorweight and size were measured, and tissue specimen frozen in O.C.T.compound. Tissue sections were stained with primary antibodies anddetected using Alexa Fluor dye-conjugated secondary antibodies.Microphotographs were obtained using a Zeiss laser scanning microscope(LSM 510). In addition, single cell suspension from tumor masses wereused to sort the BDEneu cells with RFP fluorescence. MiR-195 levels inmiR-195 treated rats and NSM treated controls were measured via RT-PCR.

Kaplan-Meyer Survival Curves

In a follow up animal in vivo experiment, 20 CCA rats were randomized ina control group of 9 CCA rats treated with NSM and a treatment group of11 rats, treated with miR-195 mimic, both starting from Day 15. Animalswere monitored daily and the date of death of each rat recorded and thedata incorporated into a Kaplan-Meyer curve. Data were analyzed with thelog-rank (Mantel-Cox) test.

Detection of the Location of EVs in CCA Mass of Liver

Isolated EVs from LX2-pCDH1-EF1-mCherry-TSG101-IRES-GFP cells wereinjected into tail veins of CCA rats. After 24 hours the rats weresacrificed and tissue specimen of the tumor mass were frozen in O.C.T.compound. Tissue sections were stained with primary antibodies againstmCherry (cat #632496 Clontech, Mountain View, Calif.) and alpha-SMA (cat# A2547, Sigma-Aldrich, St. Louis, Mo.) to detect the injected EVs andto measure the degree of the fibrotic change in the tumor mass.Furthermore, BDEneu cells infected withpMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE lentiviral construct were injectedinto rat livers as described above to generate the CCA model, and after20 days, EVs transfected with Cre plasmid were administered to the ratsvia tail veil injections. 4-6 days later, rats were sacrificed and tumorsections obtained as described above. Cells that switched color fromdsRed to EGFP indicate BDEneu tumor cells that have taken up EVs loadedwith Cre-recombinase plasmid.

Proliferation and Apoptosis Measurement In Vivo

Tumor mass specimens were embedded in paraffin, sections were stainedwith primary antibody Ki67 (cat #550609,BD San Jose, Calif.), caspase 3(cat #9661S, Cell Signaling Technology, Dancers, Mass.), alpha-SMA andTUNEL in situ cell death fluorescein (Sigma-Aldrich, St. Louis, Mo.) todetermine the levels of proliferation, apoptosis, as well as fibroticinfiltrate. Image J was used to identify the florescence intensity.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is: 1-4. (canceled)
 5. An extracellular vesicle isolatedfrom a cancer associated fibroblast (CAF), wherein the vesicle comprisesa small molecule, and wherein the extracellular vesicle selectivelytargets a cancer cell.
 6. (canceled)
 7. The extracellular vesicle ofclaim 5, wherein the small molecule is loaded into the cell orextracellular vesicle ex vivo.
 8. The extracellular vesicle of claim 5,further comprising a recombinant polypeptide or polynucleotide that isheterologously expressed in the CAF or is loaded into the cell orextracellular vesicle ex vivo.
 9. The extracellular vesicle of claim 8,wherein the recombinant polynucleotide is a microRNA.
 10. Theextracellular vesicle of claim 9, wherein the microRNA is miR-195,miR-126, or miR-192.
 11. The extracellular vesicle of claim 5, whereinthe small molecule is a lipid or other hydrophobic small molecule. 12.The extracellular vesicle of claim 5, wherein the small molecule isdoxorubicin, cisplatin, or phosphatidyl ethanolamine.
 13. Theextracellular vesicle of claim 12, wherein the phosphatidyl ethanolamineis derivatized with an agent selected from the group consisting ofrhodamine, fluorescein, biotin, streptavidin, a small molecule, apolynucleotide, and a polypeptide.
 14. The extracellular vesicle ofclaim 5, wherein the small molecule can be used for imaging purposes.15-25. (canceled)
 26. The extracellular vesicle of claim 5, wherein thevesicle expresses increased levels of one or more markers selected fromthe group consisting of alpha-SMA, Collagen, Vimentin (FSP-1), S100,Metalloproteinases, NG2, PDGFR-B, SDF1/CXCL12, CD34, Fibroblastactivation protein (FAP), FSP-1, CD31, Thy-1, and Gremlin, and/orexpresses reduced levels of laminin.
 27. (canceled)
 28. Theextracellular vesicle of claim 5, wherein the CAF is derived from afibroblast cultured for at least 1-14 days in the presence of a cancercell or in the presence of conditioned media derived from a cancer cellculture. 29-32. (canceled)
 33. A method for obtaining the extracellularvesicle of claim 5, the method comprising culturing a fibroblast orstromal cell in conditioned media obtained from a cancer cell culture,and isolating extracellular vesicles from the media. 34-29. (canceled)40. An extracellular vesicle produced according to the method of claim33.
 41. A pharmaceutical composition comprising the extracellularvesicle of claim
 5. 42. A method of delivering a small molecule to acell, the method comprising contacting the cell with the extracellularvesicle of claim 5, thereby delivering the small molecule agent to thecell.
 43. (canceled)
 44. The method of claim 46, wherein the cancer isbreast cancer, pancreatic cancer, glioblastoma, melanoma, lung cancer,ovarian cancer, or liver cancer.
 45. A method of altering geneexpression in a cell, the method comprising contacting the cell with theextracellular vesicle of claim
 5. 46. A method for treating cancer in asubject comprising administering to the subject a pharmaceuticalcomposition comprising an effective amount of the extracellular vesicleof claim
 5. 47. The method of claim 46, wherein the cancer ischolangiocarcinoma, hepatocellular carcinoma, or hepatoma.
 48. Anextracellular vesicle isolated from a cancer associated fibroblast(CAF), wherein the extracellular vesicle comprises a small molecule anda heterologous polynucleotide comprising miR-195, miR-126, or miR-192,and wherein the extracellular vesicle selectively targets a cancer cell.49. A method for treating cancer in a subject comprising administeringto the subject a pharmaceutical composition comprising an effectiveamount of the extracellular vesicle of claim
 48. 50. A pharmaceuticalcomposition comprising an effective amount of a first and a secondextracellular vesicle, wherein the first extracellular vesicle comprisesa small molecule and the second extracellular vesicle comprises aheterologous polynucleotide comprising miR-195, miR-126, or miR-192. 51.A method for treating cancer in a subject comprising administering tothe subject the pharmaceutical composition of claim
 50. 52. (canceled)53. A composition for imaging studies, the composition comprising theextracellular vesicle of claim 5, wherein the vesicle comprises adetectable or imaging agent.
 54. (canceled)
 55. The composition of claim54, wherein the imaging agent is a nanoparticle, magnetite,nanoparticle, paramagnetic particle, microsphere, nanosphere, and isselectively targeted to cancer cells. 56-57. (canceled)
 58. A kit fordelivering an agent to a cell the kit, wherein the agent comprises theextracellular vesicle of claim 5.